Unwanted hydrolysis or α/β-peptide bond formation: How long should the rate-limiting coupling step take?

Article abstract of DOI:10.1039/c9ra06124j

Nowadays, in Solid Phase Peptide Synthesis (SPPS), being either manual, automated, continuous flow or microwave-assisted, the reaction with various coupling reagents takes place via in situ active ester formation. In this study, the formation and stability of these key active esters were investigated with time-resolved ¹H NMR by using the common PyBOP/DIEA and HOBt/DIC coupling reagents for both α- and β-amino acids. Parallel to the amide bond formation, the hydrolysis of the α/β-active esters, a side reaction that is a considerable efficacy limiting factor, was studied. Based on the chemical nature/constitution of the active esters, three amino acid categories were determined: (i) the rapidly hydrolyzing ones (t < 6 h) with smaller (Ala) or even longer side chains (Arg) holding a large protecting group; (ii) branched amino acids (Ile, Thr) with slowly hydrolyzing (6 < t < 24 h) propensities, and (iii) non-hydrolyzing ones, such as the hard-to-couple β-amino acids or β-sugar amino acid derivatives, stable for longer times (t > 24 h) in solution. The current insight into the kinetics of this key hydrolysis side reaction serves as a guide to optimize the coupling conditions of α- and β-amino acids, thereby saving time and minimizing the amounts of reagents and amino acids to be used-all key factors of more environmentally friendly chemistry.

Full text of DOI:10.1039/c9ra06124j

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Unwanted hydrolysis or a/b-peptide bond
formation: how long should the rate-limiting
Cite this: RSC Adv., 2019, 9, 30720
coupling step take?†
a
b
a
c
Viktoria Goldschmidt G o} z,
Adrienn Nagy, Viktor Farkas,
Ern o} Keszei
´
ab
and Andras Perczel
*
´
Nowadays, in Solid Phase Peptide Synthesis (SPPS), being either manual, automated, continuous flow or
microwave-assisted, the reaction with various coupling reagents takes place via in situ active ester
formation. In this study, the formation and stability of these key active esters were investigated with
1
time-resolved H NMR by using the common PyBOP/DIEA and HOBt/DIC coupling reagents for both a-
and b-amino acids. Parallel to the amide bond formation, the hydrolysis of the a/b-active esters, a side
reaction that is a considerable efficacy limiting factor, was studied. Based on the chemical nature/
constitution of the active esters, three amino acid categories were determined: (i) the rapidly hydrolyzing
ones (t < 6 h) with smaller (Ala) or even longer side chains (Arg) holding a large protecting group; (ii)
branched amino acids (Ile, Thr) with slowly hydrolyzing (6 < t < 24 h) propensities, and (iii) non-
hydrolyzing ones, such as the hard-to-couple b-amino acids or b-sugar amino acid derivatives, stable for
longer times (t > 24 h) in solution. The current insight into the kinetics of this key hydrolysis side reaction
serves as a guide to optimize the coupling conditions of a- and b-amino acids, thereby saving time and
minimizing the amounts of reagents and amino acids to be used – all key factors of more
environmentally friendly chemistry.
Received 6th August 2019
Accepted 29th August 2019
DOI: 10.1039/c9ra06124j
rsc.li/rsc-advances
established: the activation of the carboxyl group via in situ active
ester formation is typical. As a result of a large number of
Introduction
Both polypeptides and proteins are evolutionarily ne-tuned coupling agents probed and different kind of active esters
1
6,17
linear polymers, playing key roles in almost every process of tested,
perhaps the most commonly used pair of reagent
1
7
cellular life. The need for polypeptide-based lead-compounds today is that of the HOBt/DIC, introduced 20 years ago. In
and drugs (e.g. oxytocin, insulin, GLP1 agonists) is increasing, addition, for coupling “difficult sequences” either PyBOP/DIEA
2–5
enhancing the role of polypeptides in drug discovery. Thus, or HATU/DIEA reagents were developed and used the most
18,19
the efficient synthesis of oligo- and polypeptides has been oen in combination with the basic HOBt/DIC
protocol. One
a current topic and a continuous challenge of synthetic chem- of the additional advantages of using PyBOP is to minimize or
istry since the second half of the last century. Solid phase even avoid racemization of the coupled amino acid residue. In
peptide synthesis (SPPS) became a technique developed and contrast to this, when using HATU the chance of racemization
6

ne-tuned ever since. Both the chemistry of introducing increases with the length of the coupling time and thus, gives
linkers, protecting groups, coupling agents, resins, etc. and the a time limit on the overall reaction, which should be set shorter
20
engineering at a given level of automation (microwave-assisted, than 3 h. According to the standard coupling protocols worked
7
–15
continuous-ow, etc.) have been improved.
A generalized, highly automated and optimized method for with the coupling agent before mixing with the (i ꢀ 1) amino
out for a-amino acid residues, amino acids are pre-activated
21
amide bond formation for peptide synthesis is now well acid (R–NH ) which requests about 10 minutes in general. The
2
typical coupling time of the residue (i ꢀ 1) to the i varies
between 1 to 3 hours for a-amino acids, occasionally increased
MTA-ELTE Protein Modeling Research Group, P a´ zm a´ ny P. stny. 1/A, 1117 Budapest, up to 18 hours for a difficult to couple residue. However, these
22
a
Hungary. E-mail: perczel.andras@ttk.elte.hu
“common” time limits rely on thumb-rules and common
understanding of peptide chemists, in default of the support of
quantitative and systematic kinetic studies. Some active esters
are commercially available, but usually it is made in situ during
the coupling step. Active esters are generally investigated in
details only when they are “rst described” as a new coupling
b
Laboratory of Structural Chemistry and Biology, Institute of Chemistry, E ¨o tv ¨o s Lor a´ nd
University, P a´ zm a´ ny P. stny. 1/A, 1117 Budapest, Hungary
c
Chemical Kinetics Laboratory, Institute of Chemistry, E ¨o tv ¨o s Lor a´ nd University,
P a´ zm a´ ny P. stny. 1/A, 1117 Budapest, Hungary
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c9ra06124j
1
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agent, like in the case of PyBOP for example, when the forma- amino acids. They have similarly polar or apolar character,
20
tion of the active esters was followed by TLC. In most cases the they are either protected or un-protected and have a consti-
time required for their formation was determined and the tution comprising a small or a large side chain in fact either
16
mechanism of activation and coupling was examined. Aer easy- or hard-to-couple to the adjacent amino acid residues.
the 1990s, each of the new coupling reagents was tested and a-Amino acids having either no/shorter –H (Gly), –CH (Ala)
2 3
compared to the others with respect to make peptides according or longer side chains –CH –CONH– (Asn) and –(CH ) -
3
2
23
to the literature data (e.g. ACP), however the comprehensive NHC(NH)NH– (Arg) with large protecting groups (Trt, Pbf,
analysis of the nascent active ester's stability was not checked respectively) form the rst group. a-Amino acids with b- and/
and probed. Thus, studies on the formation and stability of or g-branched side chains, such as Thr, Leu, Ile, and Val
amino acid active esters in the literature are sporadic, rare and constitute the second group. Aliphatic or cyclic b-amino acids
oen quite inconsistent. In the seminal work of Albericio and (normal and b-sugar amino acids) with a rigid structure and
co-workers, the stability of some active esters in DMF was fol- large protection form the third group. The latter two
7
lowed by HPLC. However, the active esters of the most common comprise the so-called hard-to-couple residues as well
a- and b-amino acid residues have not yet been systematically (Scheme 1).
tested. The dry DMF typically used for coupling contains a little,
To optimize the coupling of an amino acid in a specic
but a signicant amount of water (e.g. #0.03% according to manner both the active ester formation and its stability were
1
Sigma Aldrich). Furthermore, an inert atmosphere is sometimes followed by time-resolved H NMR spectroscopy. The solution
used during residue coupling, thus these traces of water mole- of the amino acid was mixed with PyBOP or HOBt in an
ꢁ
cules deeply perturbing the reaction, as besides the peptide equimolar amount in DMF-d at 25 C. As expected, the PyBOP
7
bond formation of interest, the hydrolysis of the water sensitive or HOBt and the N-protected amino acid form a stable and
active esters proceeds as a side reaction. Recently, we have unreactive mixture for 24 hours (stability checked by NMR). At
tested some commonly used coupling reagents for selected t ¼ 0 DIEA (in the case of PyBOP) or DIC (in the case of HOBt)
2
4,25
1
Fmoc-protected b-sugar amino acids, Fmoc-b-SAA-OH,
was added to initiate the active ester formation. H NMR
26
indeed hard to activate and couple. Considering the time spectra were recorded aer 10, 20, 30 and 60 minutes followed
needed for the active ester to be formed without hydrolysis for by hourly acquisition in the rst 6 hours, and nally aer 24
SAA derivatives the PyBOP/DIEA system turned out to be the hours. (Note, that in SPPS the typical coupling time is 1 or 3
best choice with respect to coupling efficacy, in parallel to hours). To perform a quantitative analysis rst, the non-
minimize or totally avoid racemization.
overlapping, characteristic resonance frequencies were
Here we present a systematic study on the kinetic details of assigned and monitored. Usually, the integrals of the selected
0
D
both making and preserving active esters from an array of both aromatic protons of the active esters (e.g. for Fmoc-Ile-OH, H
0
B
linear and cyclic, a- and b-amino acid residues with PyBOP and at 8.57 ppm or H at 8.16 ppm, Fig. 1b) or characteristic side
HOBt. As some especially b-amino acid and sugar amino acid chain protons (e.g. isopropyl group for Fmoc-Val-OH at
residues are of elevated costs and sluggish to couple, the 1.05 ppm, Fig. 1c, or H between 4.4–5.5 ppm in ESI†) were
a
optimization of these reaction conditions is far to be a luxury. considered.
Our goal was to establish a general relationship between the
The active ester is formed within 10 minutes for all a-, except
chemical nature and the molecular topology of these amino a few b-amino acids, which requested longer reaction times (e.g.
3
t
acids with respect to their ability of coupling. Most impor- Fmoc-b -Thr( Bu)-OH: 20 min, Fmoc-ACPC-OH: 540 min).
tantly, the hydrolysis of their active esters was in the focus, as However, even for these slowly reacting residues, their active
their stability in the presence of some water seems to be the esters are formed in a ratio of more than 50% aer 10 minutes:
key step and the limiting factor that inuences and limits both a ratio in principle sufficient to achieve a successful coupling
the coupling time and efficacy. In addition, the molecular (The last column of Table 1 shows the conversion of formation
excess of the amino acid needed to achieve a total coupling or hydrolysis of an active ester aer 10, 60 and 180 min). Based
efficiency (e.g. >98%) was also a key factor to be determined. If on this data the typical coupling time can be determined for
we could determine and categorize the activity and stability of each amino acid with PyBOP. For example, the active ester of
the active esters of the amino acids of different molecular Fmoc-Arg(Pbf)-OH hydrolyzed quickly (conversion of hydrolysis
topology, then peptide synthesis will be conducted in a more is 97% aer 3 hours, Table 1). Therefore, it seems enough to
time- and cost-effective way. Moreover, with these data at couple such an active ester only for a single hour, and unnec-
hand, the time requirement of the automated protocols of pre- essary to continue the same reaction for additional hours. In
activation and coupling could be ne-tuned and optimized at contrast, b-amino acids can be coupled for a longer time (e.g. 18
a partner specic manner.
hours) as their active esters were found to be stable at least for
24 hours.
In some cases, due to the presence of the residual water,
hydrolysis of the active esters was found to be quite substantial
Results and discussion
In our comprehensive analysis, the use of PyBOP/DIEA and thus, the starting amino acids were recovered completely
coupling reagent pair was studied with amino acid residues aer 24 hours (Scheme 2 and Table 1). However, when the
of different chemical constitution. These selected amino acid mixture of the solvent (DMF-d
residues represent most properties of common proteinogenic molecular sieves to remove the water content of HOBt$H
7
) and HOBt was dried on
O, no
2
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Scheme 1 Three different groups of the a- and b-amino acids studied: (i) a-amino acids (unbranched residues) have either shorter or longer side
chains with or without protecting groups, (ii) residues with b- and/or g-branched side chains and (iii) aliphatic and cyclic residues of a rigid
structure equipped of large protecting groups.
formation of active esters was observed. We do explain this 0.015%). Moreover, the actual H O content of the solvents
2
phenomenon, as most probably the absence of water destabi- based on the calculations using the kinetic model was found
lizes HOBt and thus, the lack of sufficient HOBt resulted in the higher in most cases, [H
failure of the coupling reaction. Thus, a minimum amount of indicated by the supplier [H
water is requested for active ester formation. According to the rming that DMF indeed contains a small, but signicant
2
O]calc ¼ 0.02–0.25 mM, than the value
2
O]origin ¼ 0.016–0.027 mM con-
manufacturers, the water content of the dedicated NMR amount of water. (This latter concentration was calculated
7 7
solvents (e.g. DMF-d , #0.05%, data from Eurisotop: 0.045– based on 0.03–0.05% of water content in 550 ml DMF-d in the
0.02%) is practically the same as that used during peptide NMR tube). During the current measurements, several stocks of
synthesis (#0.03% in DMF according to Carlo Erba 0.03– dried DMF-d7 were probed, 2–3 parallel experiments were
Scheme 2 The formation and hydrolysis of the active ester made from a-amino acid (Fmoc-Ile-OH) with PyBOP/DIEA coupling reagent pair. AB
1
¼
PyBOP (1); C ¼ amino acid; AC ¼ active ester; B ¼ phosphine oxide (2), D ¼ base; CD ¼ unidentified by-product, A ¼ HOBt (3). The H
0
0
0
00
00
B
D
B
a
a
D
B
A
resonance frequencies of the atoms highlighted with red (H , H , H , H , H , H , H , and H ) were used to monitor the reaction. Their changes
in intensity as a function of time were recorded and used to construct suitable kinetic models.
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1
ꢁ
Fig. 1 H NMR resonance frequency and intensity changes as a function of the time (T ¼ 25 C) during the active ester formation and hydrolysis
0
0
0
ip
1
D
B
of the three different groups of amino acids. Selected H-resonances (e.g. H , H as the aromatic ring protons of active ester or H as the side
chain protons of active ester) are used to monitor and analyze the kinetics of the reactions. (a) NMR spectra of the quickly hydrolyzed active ester
from Fmoc-Ala-OH with PyBOP, (b) NMR spectra of the slowly hydrolyzed active ester from Fmoc-Ile-OH with PyBOP, (c) NMR spectra of the
3
stable active ester from Fmoc-b -Val-OH with PyBOP, (d) the integral–time diagram of three different group of amino acids referenced to the
7
signal of DMF-d : residue of fast hydrolyzing property is reported in green, while slow hydrolysis is depicted with blue and non-hydrolyzing
residue with red.
completed for each. Additional evidence of the ongoing hydro- different. For the integral–time diagram the values were refer-
lysis was the appearance of HOBt (3) (e.g. signal at 7.62 ppm, enced to DMF-d : signal at 2.75/2.93 ppm was considered as
7
00
A
H , Fig. 1), as a by-product, identied by NMR measurements. unity (integral ¼ 1.0). In the case of “fast hydrolysis”, we found
In conclusion, if using the conventionally accepted ‘blind’ that the active ester decomposes within 6 h (t < 360 min) at
methodology and types of solvents, then the hydrolysis of the in a conversion > 90% (Fig. 1a and d/green points) and the starting
situ active ester indeed proceeds, and thus, active ester forma- Fmoc-AA-OH is regained quite substantially. For “slowly
tion and hydrolysis are two competing parallel reactions. hydrolyzing” active esters, a similar amount of decomposition
Therefore, it is important to focus and understand this side takes place between 6–24 h (1440 min: Fig. 1b and d/blue
reaction, in order to conduct the main reaction properly, the points), while the active esters of Fmoc-AA-OHs of the third
amide bond formation as effectively as possible. By measuring group remain stable: their decomposition is still limited (<5%)
and working out the kinetics of these coupled reactions, the even aer 24 h (Fig. 1c and d/red points) and might be called as
optimum time and conditions for coupling of a- and b-amino “non-hydrolyzing” residues.
acids can now be determined with condence. Thus, both
saving time and minimizing the extent of reagents and amino short side chain (e.g. Gly, Ala) or a long one (e.g. Arg, Asn)
acid derivatives necessary to use become possible. equipped even with a larger protecting group (Trt, Pbf).
(i) A fast hydrolyzing amino acid has typically either no/
For the amino acids of the above three groups, we have However, if these masked functional groups are sufficiently far
0
established quantitative differences in terms of their rate of from the reaction center (C ]O) and thus they do not interfere
1
hydrolysis. Data retrieved from the H-integrals of selected not with the backbone atoms, then they cannot act against
overlapping resonances as a function of the time (Fig. 1d) are hydrolysis. The only exception was Fmoc-Leu-OH, which –
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Table 1 Reaction times (min) and conversions (%) of the active ester formation (f) and hydrolysis (h) for the Fmoc protected a- and b-amino acids
a
of the three molecular topology groups. Conversion after 10, 60 minutes and 3 hours is shown which indicates the limit of the coupling
a
b
c
1
0 min: typical time of active ester formation; 1 and 3 hours: typical reaction time used for coupling. Active ester was decomposed. Active ester
is formed >99% conversion aer 3 hours.
unlike all the amino acids – did not form the active ester OH (Table 1). Due to the cis and trans congurations, the
within 10 minutes (Table 1). Its maximum conversion was only hydrolysis was slower for the D-galacto conguration
6
>
8% and did not change until 3 hours, but aer that, it was (1440 min, Table 1) than for the D-gluco derivative (300 min,
99% and started to hydrolyze quickly. This is the reason why Table 1) and thus it belongs to the second group due to its
it appears in the rst group, despite the fact that it has a g- characteristics regarding hydrolysis. While, those of the fur-
branch. (ii) If an amino acid has either a b- or a g-branch, the anoid ring (b-SAAs: Fmoc-RibAFU(ip)-OH and Fmoc-
hydrolysis proceeds slower due to the steric hindrance of the XylAFU(ip)-OH) are indeed no hydrolyzing, because they have
0
side chain on the C ]O. In some cases, the active esters were bicyclic, rigid structures which prevent hydrolysis. (v) Slow
t
stable for 1–3 hours, e.g. Fmoc-Thr( Bu)-OBt started to hydro- hydrolysis was observed for Fmoc-b-Ala-OH as the increased
lyze aer 3 hours. (iii) Active esters formed from b-amino acids backbone exibility presents some obstacle against a fast
are stable within the time frame of the current observation. hydrolysis.
The 6-membered H-bond pseudo ring structure gives higher
stability compared to that of 5-membered present in a-amino
acid active esters (Fig. 2). (iv) In the case of b-sugar amino Kinetic analysis
2
7
acids, the exibility of the pyranoside ring causes the
hydrolysis of Fmoc-GlcAPU(Me)-OH and Fmoc-GalAPU(Me)-
To determine kinetic parameters as e.g. the half-life and starting
concentration of Fmoc-AA-OH active esters, a kinetic analysis of
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As the hydrolysis follows second-order kinetics, the half-life
(t1/2) of active esters depends on the initial concentration of
water in the reaction mixture as follows:
ꢀ
ꢁ
1
c
ester;0
t
1=2 ¼ k
ln 2 ꢀ
(1)
hydrolysis ꢂ ðcwater;0 ꢀ cester;0
Þ
c
water;0
where khydrolysis is the rate constant of the hydrolysis, cwater,0 is
the initial concentration of water, and cester,0 that of the active
Fig. 2 The stabilizing H-bond in an active ester: (a) 5-membered H-
bonded pseudo-ring is formed in case of a-amino acids (highlighted ester. (Note that this formula is valid only if cwater,0 is greater
with green), whereas (b) an even more stable 6-membered H-bonded
pseudo ring emerges in b-amino acids (highlighted with red)
than cester,0 – as is the current case. If cester,0 exceeds cwater,0 but it
is not higher than twice the value of cester,0, then the two initial
concentrations should be ipped in both the difference and the
fraction. If cester,0 exceeds cwater,0 by more than a factor of 2, then
the active ester concentration cannot become as low as half of
the temporal evolution of the reactions was performed. The
the initial concentration, due to the hydrolysis.)
hydrolysis of the active esters proved to be a second-order reac-
tion. This was also supported by the fact that the measured
The half-lives were determined for the estimated initial
(
[H
2
O]
0
) and for xed water concentration ([H
2
O]
0
¼ 0.25 mM)
([AC]0,meas) and calculated ([AC]0,calc) initial concentration of the
also. For the latter, the [AC]
0
was also xed at 0.1 mM in order
active esters were in good agreement. However, for some active
esters, [AC]0,calc was signicantly larger than [AC]0,meas; in such
cases, the hydrolysis started before acquiring the rst measured
point (i.e. 10 min). This behavior occurred in the case of amino
acids which hydrolyze fast (group one). From the kinetic analysis,
the initial concentration of water ([H O] ) could also be esti-
mated, except for a few cases where the uncertainty of this
parameter was very large, due to the lack of enough experimental
points. In these cases, the initial concentration of water has been
that the half-lives were more comparable. According to this,
Fmoc-Leu-OH really belongs to the fast hydrolyzing amino
acids, based on khydrolysis and t1/2 values. Nevertheless, it is
interesting that Fmoc-Thr( Bu)-OH and Fmoc-Val-OH would
belong to this rst group (Table 2). It means that, if there would
t
2
0
be enough water in the solution, they would hydrolyze quickly
(t1/2,calc and khydrolysis) according to the calculation. However,
repeated measurements have always resulted in slow hydrolysis;
so it is due to the steric hindrance of b-branch in the side chain,
not the amount of water. For this reason, the half-life is less
indicative concerning the rate of the hydrolysis; the correct
comparison of the rates can be made based on the rate constant
xed to an average of the estimated water content of DMF (0.25
mM) found in reactions where this parameter could be deter-
mined at a small uncertainty.
0 2 0
Table 2 Kinetic parameters (khydrolysis, initial concentrations: [AC] and [H O] , half-lives: t1/2,calc) of the hydrolysis of active esters for group one
and two. Errors after the ꢃ signs are given as half widths of 95% confidence intervals of the parameters determined from the estimation
a
c
f
b
Hydrolysis already started before the rst measured point since [AC]0,calc > [AC]0,meas
.
[H
O] used as initial water concentration. With [AC]
Not applicable; the estimated water concentration was inferior to that of half of the active ester concentration.
2 0
O] was xed at 0.25 mM during parameter estimation.
d
e
The value not signicant, condence intervals are to large. [H
2
0
¼ 0.1 and [H O] ¼ 0.25 mM.
2
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khydrolysis of the second-order reaction. Taking this into account, the formation and hydrolysis will be the two competitive reac-
the grouping of active esters was independent of the actual tions, not the amide bond formation and hydrolysis, therefore
water concentration; therefore, large differences in the rate of aer the water runs out from the system the active ester
the hydrolysis were indeed caused by structural differences.
becomes stable.
Finally, the active ester formation with HOBt/DIC of selected
amino acids of all the groups was also measured and compared Practical considerations
with values obtained for PyBOP. The active esters are the same
(
i) Using HOBt/DIC as the coupling reagent pair, reaction time
benzotriazole derivatives in both cases, but the mechanism of
should be longer (2–3 h) then typical protocols propose, espe-
cially, if equimolar or only small molar excess of amino acid
derivatives is used.
26,28
formation is different for HOBt/DIC and PyBOP/DIEA.
Accordingly, it was not surprising that for all residues, the active
esters were formed slower (60–240 min, Table 1, ESI†); however,
the maximum conversions were also signicantly smaller (35–
(ii) Pre-activation should be considered, from 10 min up to 1
hour, to avoid an incomplete coupling and thus, sequence
errors to emerge, more prevalent perhaps for Arg. This was
tested for shorter peptides (e.g. EEEAVRLYIQWLK) with
73%). Nevertheless, this conversion is enough for coupling if 3
equivalent is used. Interestingly, the members of each group
behaved in the opposite way than with the former coupling
agents: (i) the slowly hydrolyzing Fmoc-Val-OBt was formed
within 240 minutes but found stable for a long time (24 h). (ii)
On the contrary, the previously stable Fmoc-RibAFU(ip)-OBt
made from PyBOP turned out to hydrolyze faster to result in
the starting b-SAA just aer 1 hour! (iii) The fast hydrolyzing
Fmoc-GlcAPU(Me)-OH was found also stable for 24 hours just
like the Val residue. It means that if an active ester forms slowly,
29
continuous ow SPPS. We found for example, that without
pre-activation of these amino acids, deletion of Arg and Asp was
observed. Note that both belong to the rst group based on their
molecular topology.
(
iii) Our current data support the use of PyBOP/DIEA, as
faster and more complete coupling reactions were monitored
for all types of residues. The use of PyBOP/DIEA is especially
recommended for non-commercial, oen inaccessible and
Fig. 3 Classification of the proteinogenic a- and selected b-amino acids based on their molecular topology. Fast hydrolyzing ones are high-
lighted green, slow hydrolyzing ones with blue and no-hydrolyzing ones with red.
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thus expensive amino acids (special protected a-amino of time (e.g. <1 h). Active esters of amino acids having a b- and/or g-
acids: e.g. Fmoc-Lys(Dde)-OH; b-amino acids or sugar amino branch in the side chain (e.g. Leu, Thr) hydrolyze much slower,
acids).
iv) For fast hydrolyzing amino acid residues (elements of the coupled for a longer time (6–18 h) to achieve a more complete
rst group) a short coupling time (t < 1 hour) is recommended to be amide bond formation. Nevertheless, some amino acids, usually b-,
implemented. Using pre-activation (continuous ow SPPS protocol) were found to be resistant against hydrolysis for 24 hours or longer
between from 6 to 24 hours, and therefore – if needed – they can be
(

29
worked out for small peptides (e.g. IFDPETGTWI), couplings were (e.g. ACPC, Fmoc-RibAFU(ip)-OH). Fortunately, these are typically
unsuccessful due to the poor stability of the active esters. hard-to-couple amino acids, thus they can be coupled for a longer
v) In the case of difficult peptide sequences, aer 3 hours of time than the standard coupling protocol prescribes; even over-
(
coupling time, we do recommend to complete a second night coupling could be set up, as their active esters are stable
coupling cycle, especially for expensive amino acids, by adding enough to resist during such an extension of the reaction time. The
additional PyBOP to the coupling mixture directly. This was high stability of b-amino acids comes from the six-membered H-
tested for the sequence –SGXGD– (X: -RibAFU(ip)-). Aer 3 hours bond pseudo-ring, which hinders both hydrolysis and coupling.
of coupling time, with additional 3 molar equivalents of PyBOP To give a quantitative account of the true nature of the hydrolysis,
the coupling efficacy has increased by 20%.
a kinetic study was performed to estimate both the initial active
(vi) Slowly hydrolyzing amino acids (residues of the second ester and water concentrations, along with the rate constant of the
group) are recommended to be coupled for longer time: e.g. 6 < hydrolysis. The results indicate that the different hydrolysis
tcoupling < 18 hours (overnight coupling). Adding (optional) capacity of the amino acids does not depend on the concentration
surplus of PyBOP/DIEA to the coupling mixture aer 6 hours of water; it is rather caused by their diverse structure.
could be advantageous.
(
vii) A given excess of the coupling reagent and the amino
Experimental section
acid will typically compensate the consequences of the hydro-
1
lysis and assure coupling. This was shown by the good coupling Analytical data for all compounds: H NMR spectra and gures
efficacy of Fmoc-RibAFU(ip)-OH with HOBt/DIC in a –GXXG– of active ester formation of all amino acids can be found in ESI,†
short chimera peptide by using 3 equivalent of sugar amino in the online version.
26
acid (despite of the fact, that this active ester is unstable).
However, in the case of expensive amino acids (e.g. b-amino Reagents and instrumentations
acids, special protected a-amino acids) it may be worth to
Reagents, materials and solvents were obtained from Sigma
reduce the excess of amino acids or to use other coupling
reagents (e.g. PyAOP) or solvent (e.g. NMP).
Aldrich, Irish Biotech GMBH or Eurisotop. b-Sugar amino acids
24,25
were synthetized in our laboratory based on our previous works.
(viii) Proteinogenic amino acids are thus proposed to couple
as follows:
NMR measurements
-
in the case of Gly, Ala, Asn, Phe, Tyr, Cys, Met, Gln, Asp, Glu,
1
H NMR experiments were performed at 298–300 K on Bruker
Arg and Lys (members of the rst group) coupling should be
conducted as described in point iv.
Avance DRX 250 MHz spectrometer equipped with 5 mm SB
dual probe with z-gradient, operating at 250.13 MHz for H and/
or on a Bruker Avance III 700 spectrometer operating at 700.05
MHz using a Prodigy TCI H&F-C/N-D, z-gradient probe head.
1
-
amino acids as Val, Pro, Ser, Thr, Leu, Trp, His and Ile
members of the second group) a successful coupling requires
pre-activation or longer coupling time (3 h).
nally, b-amino acid and b-sugar amino acid residues
(
Spectra were recorded in DMF-d using the solvent residual
7
-
1
peaks as the H internal reference: 2.75, 2.93 and 8.03 ppm. The
constitute the third group, where a much longer coupling time
of 3–18 hours is needed (Fig. 3).
sample concentrations ranged from 10 to 20 mM (Table 2).
Spectra evaluation was completed within TopSpin 3.5 soware.
Conclusion
Kinetic analysis
A very important accompanying side reaction of peptide bond Kinetic parameter estimation was based on the integral of
formation is quantitatively analyzed here. We have determined selective NMR signals considered to be proportional to the
based on the formation and stability of the active esters of various concentration of the relevant species. The mechanism taken into
a- and b-amino acids with PyBOP/DIEA or HOBt/DIC key kinetic account is the one shown on top of Scheme 2, but only the
1
parameters, by using time-resolved H NMR spectroscopy. It hydrolysis step was modeled. For the parameter estimation, the
turned out that these species are able to hydrolyze parallel to the COPASI 4.16 (Build 104) Biochemical System Simulator soware
amide bond formation due to traces of water inherently present. (http://copasi.org/) was used, with the parameter estimation
Based on the kinetic data established for hydrolysis, three different option of the Levenberg–Marquardt method. The result of the
groups of amino acid residues were identied. Amino acids having estimation procedure did not depend on the choice of the initial
a small (e.g. G, A) or long side chain and equipped with a large parameters within a large interval, thus there was one stable
protecting group (Asn, Arg), but placed far from the reaction center optimum for the t of the model only. Condence interval half-
hydrolyzes quickly: within less than 6 hours. This fact indicates widths were calculated from the estimated standard deviations
that it is necessary to couple these amino acids within a short range based on the Student distribution with n ꢀ p degrees of freedom,
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Paper
where n is the number of data in the concentration vs. time
measurements and p is the number of parameters estimated.
Usually, three parameters were estimated: the rate constant of the
hydrolysis khydrolysis and the initial concentrations of the active
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0
and that of the water [H
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. In some cases, the esti-
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estimated concentration in other cases.
6
7
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Compliance with ethical standards
This article does not contain any studies with human partici-
pants or animals performed by any of the authors.
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The authors declare that they have no conict of interest.
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1
1
1
1
2 G. B. Fields and R. L. Noble, Int. J. Pept. Protein Res., 1990, 35,
Fmoc-
Methyl 2,3-di-O-benzyl-N-(9-
161–214.
GlcAPU(Me)-OH uorenylmethoxy-carbonyl)-4-amino-4-deoxy-
a-D-glucopyranoside uronic acid
Fmoc-
GalAPU(Me)-
OH
Fmoc-
3 J. M. Collins, K. A. Porter, S. K. Singh and G. S. Vanier, Org.
Lett., 2014, 16, 940–943.
Methyl 2,3-di-O-benzyl-N-(9-
uorenylmethoxy-carbonyl)-4-amino-4-deoxy-
a-D-galactopyranoside uronic acid
4 I. M. M ´a ndity, B. Olasz, S. B. Otvos and F. F u¨ lop,
¨
¨
¨
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1,2-O-isopropylidene-N-(9-uorenylmethoxy-
RibAFU(ip)-OH carbonyl)-3-amino-3-deoxy-a-D-
ribofuranuronic acid
1,2-O-isopropylidene-N-(9-uorenylmethoxy-
1
1
6 E. Valeur and M. Bradley, Chem. Soc. Rev., 2009, 38, 606–631.
7 A. El-Faham and F. Albericio, Chem. Rev., 2011, 111(10),
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XylAFU(ip)-OH carbonyl)-3-amino-3-deoxy-a-D-
xylofuranuronic acid
Fmoc-ACPC-OH N-(9-uorenylmethoxy-carbonyl)-2-amino-
cyclopentanecarboxylic acid
Fmoc-ACHC-
OH
6557–6602.
18 W. Koenig and R. Geiger, Chem. Ber., 1970, 103, 788–798.
19 W. Koenig and R. Geiger, Chem. Ber., 1970, 103, 2024–2033.
20 J. Coste, D. LeNguyen and B. Castro, Tetrahedron Lett., 1990,
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N-(9-uorenylmethoxy-carbonyl)-2-amino-
cyclohexanecarboxylic acid
Fmoc-AA-OH
Fmoc protected a-amino acid
21 M. Amblard, J.-A. Fehrentz, J. Martinez and G. Subra, Mol.
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2 M. Goodman, Synthesis of peptides and peptidomimetics,
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2
Acknowledgements
The authors wish to thank D ´a niel Horv ´a th for helping 700 MHz 24 A. Nagy, B. Csord ´a s, V. Zsoldos-M ´a dy, I. Pint ´e r, V. Farkas and
NMR measurements and Tam ´a s Martinek for Fmoc-ACPC-OH,
A. Perczel, Amino Acids, 2017, 49, 223–240.
Fmoc-ACHC-OH and linear b-amino acids. These research 25 V. Goldschmidt G o} z, I. Pint ´e r, V. Harmat and A. Perczel, Eur.
projects were supported by the European Union and the State of
J. Org. Chem., 2018, 3, 355–361.
Hungary and co-nanced by the European Regional Development 26 A. Nagy, V. Goldschmidt G o} z, I. Pint ´e r, V. Farkas and
Fund (VEKOP-2.3.2-16-2017-00014). This paper was supported by
A. Perczel, Amino Acids, 2019, 51, 669–678.
the J ´a nos Bolyai Research Scholarship of the Hungarian Academy 27 H. B. Mayes, L. J. Broadbelt and G. T. Beckham, J. Am. Chem.
of Sciences (V. Farkas) and MedInProt Grant Facilitating Access to
Soc., 2014, 136, 1008–1022.
Instruments from the Hungarian Academy of Sciences.
28 C. A. G. N. Montalbetti and V. Falque, Tetrahedron, 2005, 61,
10827–10852.
2
9 V. Farkas, K. Ferentzi, K. Horv ´a ti, A. Perczel, 2019, under
preparation.
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30728 | RSC Adv., 2019, 9, 30720–30728
This journal is © The Royal Society of Chemistry 2019