Cis-Trans Conformational Analysis of δ-Azaproline in Peptides

Article abstract of DOI:10.1021/acs.joc.5b01668

The cis-trans isomerization and conformer specificity of δ-azaproline and its carbamate-protected form in linear and cyclic peptides were investigated using NMR and α-chymotrypsin assay. Comparisons of the chemical shift value of the α-hydrogen in each case of δ-azaproline-containing peptides with conformer-specific locked diketopiperazines reveal the fact that an upfield chemical shift value corresponds to cis conformer and a downfield value corresponds to a trans conformer. δ-Azaproline adopts cis-conformation in simple amides, dipeptides, and tripeptides whereas its carbamate-protected form adopts trans-conformation. In the case of longer, linear or cyclic peptides, vice versa results are obtained. Interestingly, in all these peptides exclusively one conformer, either cis or trans, is stabilized. This cis-trans isomerization is independent of both temperature and solvents; only the δ-nitrogen protecting group plays key role in the isomerization. δ-Azaproline is conformer-specific in either of its protected or deprotected forms, which is a unique property of this proline. Unlike other covalently modified proline surrogates, this isomerization of δ-azaproline can be tuned easily by a protecting group. The mechanism of cis-trans isomerization of δ-azaproline during deprotection and reprotection is supported by theoretical calculations.

Full text of DOI:10.1021/acs.joc.5b01668

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Article
Cis - Trans Conformational Analysis of delta – Azaproline in Peptides
Indranil Duttagupta, Debojyoti Misra, Sourav Bhunya, Ankan Paul, and Surajit Sinha
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.5b01668 • Publication Date (Web): 06 Oct 2015
Downloaded from http://pubs.acs.org on October 6, 2015
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Cis - Trans Conformational Analysis of δ – Azaproline in Peptides
†
†
‡
‡
Indranil Duttagupta, Debojyoti Misra, Sourav Bhunya, Ankan Paul and Surajit
,
†
Sinha*
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†
Department of Organic Chemistry, Indian Association for the Cultivation of Science, Jadavpur,
Kolkata 700 032, India, *ocss5@iacs.res.in
‡
Raman Centre for Atomic, Optical and Molecular Physics, Indian Association for the
Cultivation of Science, Jadavpur, Kolkata 700 032, India
The cisꢀtrans isomerisation and conformer specificity of δꢀazaproline and its carbamate protected
form in linear and cyclic peptides were investigated using NMR and αꢀchymotrypsin assay.
Comparisons of chemical shift value of the α – hydrogen in each case of δ–azaprolineꢀcontaining
peptides with conformer specific locked diketopiperazines reveal the fact that an upfield
chemical shift value corresponds to cis conformer and a downfield value corresponds to a trans
conformer. δꢀAzaproline adopts cisꢀconformation in simple amides, dipeptides and tripeptides
whereas its carbamate protected form adopts transꢀconformation. In the case of longer, linear or
cyclic peptides, viceꢀversa results are obtained. Interestingly, in all these peptides exclusively
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one conformer either cis or trans is stabilized. This cisꢀtrans isomerisation is independent on
both temperature and solvents, only the δ ꢀ nitrogen protecting group plays key role in the
isomerization. δꢀAzaproline is conformer specific in either of its protected or deprotected forms,
which is a unique property of this proline. Unlike other covalently modified proline surrogates,
this isomerization of δꢀazaproline can be tuned easily by a protecting group. Mechanism of cisꢀ
trans isomerisation of δꢀazaproline during deprotection and reprotection is supported by
theoretical calculations.
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INTRODUCTION
Cyclic αꢀhydrazino acids (1) (Figure 1) have emerged as one of the most promising non ꢀ
proteinogenic amino acid candidate and they may find use as proline surrogates in order to
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improve peptide pharmacokinetics and/or bioavailability. However before being considered as a
proline substitute, their effect on the structure of peptides needs to be thoroughly investigated,
since the function of peptides and proteins largely depend on their structure.
Figure 1. General structure of cyclic α ꢀ hydrazino acids
In protein structures, due to significant delocalization of the lone pair of electrons on the nitrogen
atom the peptide bond attains a partial double bond character. The partial double bond renders
the amide group planar, allowing it to either adopt a cis or trans conformation. These bonds are
free to adopt either of the conformations in the unfolded state, but are only allowed to adopt the
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energetically more favorable, trans conformation in folded state.
Proline is unique among other naturally occurring amino acid, being an imino acid proline
residue restricts the conformational space of the peptide chain. However Xaa ꢀ Pro peptide
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groups tend to have a roughly 3:1 cis and trans ratio, apparently because the symmetry between
α
δ
the C and C atoms of proline makes both the conformers nearly equal in energy. Consequently
any change in α and δ positions will be reflected in the cis ꢀ trans ratio as the symmetry will be
disrupted. Because of this unique conformational property, proline plays an important role in
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aꢀf
structural and biological properties of peptides and proteins. Loss of symmetry owing to any
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gꢀi
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substitution at the α or δ position of proline has effects on the structure of peptides as have
been investigated by several groups. In most cases conformational studies of peptides or protein
folding is hampered by the heterogeneity of the XaaꢀPro peptide group due to the presence of
cis/trans mixtures. In order to eliminate complications arising from the cis/trans isomerisation of
proline peptide groups, scientific community has constantly been on the lookout for either a cis
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,4
or a trans stabilizing proline analogues or surrogates (diminishing or enlarging the pyrrolidine
4k
ring size). In this context, the conformational behavior of pyrazolidines as proline equivalent in
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peptides has been extensively explored by Marshall and coworkers. To some extent
pyrazolidines resembled proline and could stabilize cis conformer in organic solvents and in
solid phase, however it loses its conformer specificity in aqueous solutions. Taking a cue from
these studies we contemplated the use of δ – azaproline 1 as proline surrogate in order to achieve
conformer specificity.
δ ꢀ Azaproline 1, a five membered cyclic α ꢀ hydrazino acid, closely resembles proline in
structure and ring size, only difference being the nitrogen atom at the delta position instead of
carbon. Herein we wish to report the effect of δ ꢀ azaproline and a carbamate protecting group at
δ ꢀ nitrogen on cis ꢀ trans conformation of δ ꢀ azaproline containing linear and cyclic peptides.
RESULTS AND DISCUSSION
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Model Study. The cis ꢀ trans conformation of δ – azaproline containing peptide is shown in
Figure 2, which is defined by the spatial arrangement of peptide chains, either lying syn or anti to
each other is termed as the cis or trans conformer, respectively.
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Figure 2. (a) Cis ꢀ Trans isomerisation in δ ꢀ azaproline, (b) Relative positions in δ ꢀ azaproline.
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Chemical shift values of α ꢀ Hydrogen in H ꢀ NMR was our preferred tool for the identification
of cis and trans conformers of δ – azaproline and its peptides. Initially we investigated the
chemical shift of α ꢀ H corresponding to the cis ꢀ locked and trans ꢀ locked diketopiperazines of δ
– azaproline and accordingly, the compounds were synthesized. Compound 2 was treated with
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20% TFA in DCM followed by selective benzyloxy carbonyl protection of δꢀnitrogen 3.
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Compound 3 was heated with FmocꢀValꢀCl and AgCN in benzene to yield 4, which on Fmoc
deprotection and heating in the same pot yielded the diketopiperazine 5. The compound 5 is
locked in cis conformation (Scheme 1).
Scheme 1. Synthesis of cisꢀlocked diketopiperazine 5
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Similarly, we attempted to synthesis trans ꢀ locked diketopiperazine from 4, but isolated 5 as the
sole product. Accordingly, scheme 2 was followed to synthesize the desired product. Compound
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a was hydrolyzed by LiOH in CaCl medium followed by peptide coupling with Lꢀ
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8b
phenylalanine methyl ester to yield 6. Fmoc deprotection and in situ heating yielded the
diketopiperazine 7. The diketopiperazine 7 is locked in trans conformation (Scheme 2).
Scheme 2. Synthesis of transꢀlocked diketopiperazine 7
In order to establish the electronic effect of the carbonyl group adjoining the δ ꢀ nitrogen on the
αꢀH ppm value, compound 5 was hydrogenated in order to remove the Cbz protection to yield 8
(Scheme 3).
Scheme 3. Synthesis of deprotected cisꢀlocked diketopiperazine 8
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NMR ( H, HMQC, and COSY supporting info) studies on compounds 5, 7 and 8 were performed
to establish the signals corresponding to αꢀhydrogen, the observation were as follows.
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Compound 5. The αꢀhydrogen signal of diketopiperazine 5 was identified at 4.25. Being locked
in cis conformation, this value (ppm) was taken as the standard region for cis conformers of δ –
azaprolineꢀcontaining peptides (Figure 3).
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Figure 3. H NMR and HMQC of compound 5a
Compound 7. The α ꢀ hydrogen signal of diketopiperazine 7a was identified at 4.86. Being
locked in trans conformation, this value (ppm) was taken as the standard region for trans
conformers of δ ꢀazaprolineꢀcontaining peptides (Figure 4).
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Figure 4. H NMR and HMQC of compound 7
Compound 8. The α ꢀ hydrogen signal of the deprotected diketopiperazine 8a was identified at
4.27. Being locked in cis conformation, this value (ppm) was taken as the standard region for
deprotected cis conformers of δ ꢀ azaprolineꢀcontaining peptides (Figure 5).
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Figure 5. H NMR and HMQC of compound 8a
(
For detailed NMR spectra see supporting information)
In conclusion the cis conformer generally can be identified by upfield α ꢀ hydrogen signal, the
trans conformer by a downfield α ꢀ hydrogen signal, also the protecting groups at δ ꢀ nitrogen
has no notable electronic effect on the chemical shift of α ꢀ hydrogen.
Picking up the required information from the above experiments, we proceeded to determine the
actual inclination of protected and unprotected δꢀazaproline towards cis or trans conformation.
To establish the cis ꢀ trans conformation in δꢀazaproline, we considered a model study based on
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previous literature reports on N ꢀ acetyl proline methyl ester. The H NMR of N – acetyl proline
methyl ester showed the presence of two isomers in roughly 3:1 ratio (78:22) i.e. 78% trans and
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2% cis characterized by the α ꢀ H peaks at 4.33 (dd, J = 8.7, 3.6 Hz, 0.78H) corresponding to
trans and at 4.27 (dd, J = 8.6, 2.8 Hz, 0.22H) for cis conformers, respectively (see Supporting
Information).
Analogous azaproline based substrate 9 was prepared by acetylating 3 with acetyl chloride and
AgCN in benzene (Scheme 4).
Scheme 4. Synthesis of model substrate 9
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H NMR and HMQC studies on 9 indicated the presence of only one conformer (Figure 6).
Comparing those with the chemical shift values from previous section it can be said that Cbz
protected δꢀazaproline exists exclusively in the trans conformation.
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Figure 6. H NMR and HMQC of 9
We have synthesized another azaproline derivative 10 with a photo labile protecting group. Such
protecting groups are known to cleave in the presence of light and this strategy could be useful in
biological research. Compound 2 was converted to 11 by selectively protecting with veteroxy
chloroformate followed by acetylation (Scheme 5).
Scheme 5. Synthesis of N ꢀ Voc protected model substrate 11
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H and 2D NMR studies of 11 again illustrated the presence of trans conformer exclusively,
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Figure 7. H NMR and HMQC of 11
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similar to that of compound 9 (Figure 7). The role of electronic parameters (presence of NO ) on
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To spot the effect of deprotection, compound 9 was subjected to hydrogenation condition to
yield 12 (Scheme 6). NMR analysis of 12 showed the presence of only cis conformer, indicating
a crossover from trans conformation during the deprotection (Figure 8).
Scheme 6. Synthesis of deprotected model substrate 12
The effect of electronic factor on the chemical shift value was again ruled out by comparing with
compound 8.
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Figure 8. H NMR and HMQC of 12
The possible preference for cis conformation of the deprotected form (12) was initially thought
to be due to the formation of an intramolecular Hꢀbonding between the hydrogen attached to δꢀ
nitrogen and the adjacent oxygen of N ꢀ acetyl (highlighted in red) but rapid deuterium exchange
with D O suggested otherwise (Figure 9). Since, a proton which is part of an intramolecular
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hydrogen bond is expected to exchange slowly if at all.
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Figure 9. H ꢀ D exchange experiment on 12
After this investigation, we tried to establish the temperature dependence of cis ꢀ trans
isomerisation between the two conformers. Accordingly high temperature NMR studies on both
the compounds 9 and 12 were performed. The compounds were dissolved in DMSOꢀd6 and
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proton NMR was recorded at an interval of 10 C (25 to 105 C).
Both the Cbz protected model substrate 9 and deprotected substrate 12 did not undergo any cis ꢀ
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trans isomerisation in the temperature range between 25 to 105 C (Figure 10).
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Figure 10. (a) Variable temperature NMR of 9, (b) Variable temperature NMR of 12, (Values in
the spectra denotes the temperature)
Apart from expected shift the change of solvent (CDCl to DMSOꢀd6) also had no effect on the
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investigation.
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These investigations also begs the question about the fate of 12 on reinstallation of a protecting
group, consequently it was subjected to standard Cbz protection condition (DMAP, Et N and
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CbzCl) only to recover the starting material. Harsher conditions (AgCN, CbzCl and 60 C)
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yielded protected δ ꢀ azaproline which was identical to previously synthesized 9 in all aspects
(Scheme 7).
Scheme 7. Reprotection of model substrate 12
From the above model studies it was concluded that the δ–azaproline, in simple amides
exclusively adopts a trans conformation in its protected form and cis conformation in its native
form. These conformations resist interconversion even at elevated temperatures, only protection
or deprotection seems to bring about the conformational changes.
At this point to gain an insight into the mechanism of isomerization during deprotection or
reprotection we turned towards theoretical calculations.
I. Computational Details:
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We have optimized each intermediates and transition states using ωB97XꢀD functional with
Pople's 6ꢀ31++g(d,p) basis set on each atom except Ag atom. For Ag atom we have used LANL2
pseudopotential for core electrons and LANL2DZ basis set for valance electrons. All
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optimization were done in presence of continuum with the help of SMD solvent model and
appropriate dielectric constant of solvent. For deprotection of 9t (t stands for trans) using Pd/H2
we have used DMSO as solvent and for protection of 12c (c stands for cis) using AgCN and
CbzCl we have used benzene as solvent. Determination of entropy of a species in solution phase
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is difficult to compute, so an approximation proposed by Wertz to compute solution phase
entropies from gas phase entropies based on experimental results was used. This approximation
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is frequently employed for evaluating free energies by static quantum chemical calculations.
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We have calculated and incorporated the decrease of entropy in solution phase by reducing gas
phase entropy to 0.5 times its actual value. All theoretical calculations are done using standard
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Gaussian09 quantum chemical package.
II. Mechanism of deprotection of Cbz protected amide using Pd/H2:
Optimized structure of 9t (shown in Figure 11) shows a strong CHꢀπ interaction between
methyl CH of amide bond present nearest to the chiral center and the benzene ring present in the
benzyl group which is used for protection of the amine. In the optimized structure of 9t, amide
Figure 11. Optimized structures of 9t, 9t_1, Ts1', 9t_2'. Bond distances are shown in Å. C, N
and O are shown in grey, blue, red color. H atoms are shown in grey also but they have smaller
van der wall radii compared to C atom. This color coding is maintained throughout this section.
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CꢀN bond distance is 1.37 Å. It is well known that during deprotection of Cbz protected amine by
Pd/H , a carbamic acid intermediate is generated which undergoes decarboxylation to produce
2
1
6
the deprotected amine. We have optimized the in situ generated carbamic acid, 9t_1 (shown in
Figure 11) from 9t. Optimized structure of 9t_1 shows hydrogen bonding between the proton of
carbamic acid with the amide nitrogen and the carbonyl group of the substituent present in the
chiral carbon. Proton of carboxylic acid in 9t_1 can be transferred through a four member
transition state Ts1' (shown in Figure 11) to the nitrogen atom to produce 9t_2' (Shown in
Figure 11) with free energy activation barrier of 37.3 kcal/mol. This barrier height is
10
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Figure 12. Optimized structure of Ts1, 9t_2, 9t_3, 9t_4, Ts2' and 9t_5'. Bond distances are
shown in Å.
insurmountable in the reaction conditions. Also if this decarboxylation happens we would get a
trans isomer of 12. But our experimental results show that cis isomer of 12 is obtained
selectively. Optimized structure of 9t_1 (shown in Figure 11) shows that CꢀN amide bond length
is 1.38 Å, which is longer compared to 9t (shown in Figure 11). So we looked into the possibility
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of the rotation of the amide bond to produce cis isomer of carbamic acid, 9t_2 (shown in Figure
#
1
2). Our theoretical computation predicts very low free energy activation barrier (ΔG = 15.3
kcal/mol) for the amide bond rotation via Ts1 and the reason behind this low activation barrier is
the stabilization provided by the proton of carboxylic acid to the lone pair of nitrogen during
rotation. As a result of amide bond rotation 9t_3 is obtained and it consists of a strong hydrogen
bonding interaction between carbonyl oxygen of the amide group and the proton of the
carboxylic acid. Starting with 9t_3, an intramolecular proton transfer from carboxylic acid to the
carbonyl oxygen atom of the adjacent amide group yielded 9t_4 (shown in Figure 12).
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9
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Figure 13. Optimized structure of Ts2, 9t_5, Ts3 and 12c. Bond distances are shown in Å.
The optimized structure of 9t_4 shows that the amide CꢀN bond length is shortened and the
adjacent carbonyl CꢀO bond length has increased appreciably than that of 9t_3, suggesting the
lone pair donation tothe π* orbital of the protonated carbonyl group. We did not get any
transition state for the 9t_3 to 9t_4 transformation. We have performed a relaxed scan to locate
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the transition state for the above mentioned process but no transition state for this proton transfer
process could be identified. Hydrogen bonding interaction between proton of the imidic acid
form of protonated amide and the lone pair of adjacent nitrogen atom in 9t_4 (shown in Figure
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1
2), clearly suggests another possible intramolecular proton transfer. This proton transfer
happens via Ts2 (structure shown in Figure 13) with free energy activation barrier of 21.8
kcal/mol.
Figure 14. Relative free energy profile for decarboxylation process of carbamic acid generated
from 9t during deprotection using Pd/H2.
As a result of this proton transfer 9t_5 is formed (an exoergic process), followed by
decarboxylation of 9t_5 via Ts3 with an activation barrier of 11.0 kcal/mol leading to the
formation of 12c. Though formation of 12c is endoergic in nature, the deprotection process is
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favored as the release of CO from solution phase to gas phase creates a non equilibrium
2
situation.
Decarboxylation can also happen before intramolecular proton transfer i.e. from 9t_4 via
Ts2' (Figures 12 and 14). Since this route requires an activation energy of 35.0 kcal/mol. it is
very unlikely for the reaction to follow this path.
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Our detail theoretical investigation shows that the minimum energy pathway for
deprotection of 9t involves rotation of the amide bond which plays a crucial role in the
deprotonation of carbamic acid and subsequently facilitates the decarboxylation process. So our
theoretical calculations offer an explanation for the crossover from trans to cis isomer during
deprotection of amide (9t).
III. Mechanism for protection of amine using Cbz and AgCN:
In this section we have investigated the mechanistic details of conformational change in
the amide group during protection of 12c with AgCN and Cbz. We found that carbonyl oxygen
of the Cbz can coordinate with AgCN to produce int1 (see Figure 15). Optimized structure of
int1 shows the increase in CꢀO (present in Cbz) bond length (the bond length of C=O group of
int1 is 1.21 Å whereas the C=O bond length of Cbz is 1.19 Å) (see Figure 15). So AgCN acts as a
Lewis acid to polarize the carbonyl group facilitating subsequent nucleophilic addition by amine
12c. Formation of int1 is exothermic by 7.2 kcal/mol in terms of free energy. Nucleophilic
addition of 12c to the carbonyl group of int1 produces int2 via Ts4 with free energy activation
barrier of 3.7 kcal/mol (see Figure 15). Formation of int2 is endothermic compared to the
separated reactants (int1 and 12c) by 2.34 kcal/mol. Optimized structure of int2 shows that
amide CꢀN bond length is 1.39 Å (Figure 15), which is longer compared to 9t_1. Starting with
int2, the amide bond can rotate through Ts5 (with free energy activation barrier of 17.6 kcal) to
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attain trans form of the nucleophilic addition product, int3 (Figure 15). So this low free energy
activation barrier allows the conversion of cis to trans form of the amide during protection of 12c
by AgCN and Cbz which is observed experimentally.
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Figure 15. Optimized structures of important intermediates and transition states related to the
conformational change of the amide bond of 12c during protection using AgCN and Cbz. Bond
distances are shown in Å.
To experimentally establish and generalize this observation in tripeptides, the
1
7a
chymotrypsin coupled assay developed by Fischer et al. was used. Short model peptides of
type SucꢀValꢀΨProꢀPheꢀpNA are hydrolyzed by chymotrypsin between Phe and pNA only if
the ValꢀΨPro peptide bond is in the trans conformation (the degree of tolerance of enzymes
1
8,3a
towards proline surrogates was demonstrated by M. Mutter et.al.).
High rate of Phe ꢀ pNA
hydrolysis by enzyme is expected of the protected δꢀazaprolineꢀcontaining model peptide
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(
known as burst phase kinetics), on the other hand the deprotected δꢀazaproline containing model
peptide is not expected to undergo hydrolysis.
The azaprolineꢀcontaining model peptides for chymotrypsin studies were prepared from 4a. The
free acid 13 on treatment with oxalyl chloride followed by AgCNꢀmediated coupling with Lꢀ
PheꢀpNA provided the tripeptide 14. The compound 14 on Fmoc deprotection and subsequent N
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0
1
9
–
succinylation provided the protected model peptide 15. The tripeptide 15 was then treated
2
0
with 20% HBr in acetic acid to yield the deprotected tripeptide 16 (Scheme 8).
Scheme 8. Synthesis of chymotrypsin substrates 15 and 16.
NMR analysis of the tripeptides 15 (protected) and 16 (deprotected) showed anticipated results,
i.e. 15 adopted a trans conformation (Figure 16a) and compound 16 adopted a cis conformation
(Figure 16b).
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H
N
a
O
O
O
O N
2
NH
N
Ph
N
H
O
NCbz
10
11
12
13
14
15
16
17
18
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22
23
24
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O
15
HO
H
N
b
O
O
O
O N
NH
2
N
Ph
N
H
O
NH.HBr
O
16
HO
1
1
Figure 16. (a) H NMR and HMQC of 15, (b) H NMR and HMQC of 16
Chymotrypsin assay. The compound 15 (protected) has a trans δꢀazaproline and hence is
expected to undergo hydrolysis releasing p ꢀ nitroaniline. This was confirmed by the assay as the
reaction (release of p ꢀnitroaniline) completed within 12 seconds (Figure 17a).
Compound 16 (deprotected). Since the compound 16 has a cis δꢀazaproline, consequently it is
not expected to undergo hydrolysis in presence of chymotrypsin, which was also corroborated
with the experiment (Figure 17b).
Steep rise in case of 15 (Figure 17a) is the characteristic of a burst phase kinetics denoting the
exclusive presence of trans conformer in solution. On the other hand a flat line (Figure 17b) in
case of 16 indicates no reaction at all due to the exclusive presence of cis conformer.
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b
Figure 17. (a) Chymotrypsin assay of 15, (b) Chymotrypsin assay of 16.
After a thorough investigation of the cisꢀtrans profile of δꢀazaproline 1 in amide and tripeptides
we decided to investigate the same for a longer linear and a cyclic peptide. Accordingly a proline
2
1
containing naturally occurring cyclic pentapeptide 17, isolated from endolichenic Xylaria sp.
was chosen as a target, the linear precursor will serve the purpose of a linear model and the
compound 18 will serve as the cyclic model. The Dꢀamino acids were replaced by their
corresponding L isomers and the D isomer of δꢀazaproline instead of proline, was used for this
study (Figure 18).
Figure 18. Naturally occurring cyclic peptide 17 and our target 18.
The retrosynthetic analysis of our target 18 identified FmocꢀLꢀLeu, BocꢀLꢀIle, LꢀLeuꢀOMe, Lꢀ
ValꢀOMe and δꢀNꢀCbzꢀDꢀAzaprolineꢀOMe 3 as the required starting materials (Scheme 9).
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Scheme 9
.
Retrosynthetic analysis of target molecule 18C.
The forward synthesis commenced from the synthesis of δꢀazaprolineꢀcontaining dipeptide 21
using the previously employed silver cyanide method. The diastereomers hence obtained were
separated and the dipeptide δꢀNꢀCbzꢀDꢀazaprolineꢀOMeꢀFmocꢀLꢀLeu 21b was used for further
reactions (Scheme 10).
O
N
NHFmoc
O
N
NHFmoc
CO Me
2
Fmoc-L-Leu-Cl, AgCN
MeO C
MeO C
2
2
o
NH
NCbz
C H , 60 C, 30 min, 83%
6
6
NCbz
NCbz
21b
3
2
1a and 21b
Scheme 10. Synthesis of dipeptide 21.
Methyl ester deprotection followed by coupling with LꢀLeuꢀOMe yielded the tripeptide 22. The
compound 22 on Fmoc deprotection in piperidine followed by another coupling with BocꢀLꢀIle
yielded the NꢀCbz deprotected tetrapeptide 23.
At this point we envisaged that due to steric factors the secondary δꢀnitrogen would be unable to
take part in further peptide coupling and hence proceeded with 23. The tetrapeptide 23 on ester
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hydrolysis followed by coupling with LꢀValꢀOMe yielded the linear pentapeptide 24. Ester
hydrolysis and NꢀBoc deprotection of 24 yielded the linear precursor which was cyclized using
2
2
NaHCO and BOP in DMF to yield the cyclic peptide 18T (T stands for the trans conformer,
3
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Scheme 11).
Scheme 11. Synthesis of cyclic peptide 18T.
NMR studies of the compound 18T showed epimerization at the valine center which is indicative
of slower rate of cyclization which in turn designates the trans conformation at the δꢀazaproline
amide bond. It was confirmed by Xꢀray studies of 18T (Figure 19).
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1
Figure 19
.
XꢀRay structure, H NMR and HMQC of epimerized cyclic peptide 18T.
This might appear as a contradiction to our claim and theoretical studies in the previous section
but an analysis of the Cbz deprotection (Scheme 12) provided the answer i.e. for base mediated
deprotection of an otherwise acid labile protecting group, the trans conformation is essential and
the conversion of trans to cis becomes restricted once the deprotection takes place. This trans
conformation is carried forward till the final step and is evident from the NMR and XꢀRay
studies (Figure 19).
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0
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9
0
Scheme 12. Mechanism of Cbz deprotection of 22.
The product 25 and the byproduct 26 were identified using HRMS (Supporting info)
substantiating the above proposed mechanism. The Hꢀbonding between the NH and the carbonyl
of the Cbz increases the electrophilicity at the carbonyl carbon allowing nucleophilic piperidine
to carry out the deprotection.
From the above analysis it was hypothesized that the in situ Cbz deprotection could be arrested
by solvating the liable NH group with a polar aprotic solvent like DMF (27). Accordingly,
2
Fmoc deprotection was done in DMF using piperidine which yielded the desired Fmoc
deprotected product 28. The product 28 under EDC coupling condition in DCM yielded the Cbz
deprotected tetrapeptide 23 with a trans conformation (Scheme 13).
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Scheme 13. Addressing in situ Cbz deprotection.
The effect of reprotection at the δꢀN will be noteworthy at this point (since in the previous
section and theoretical calculations we observed that reprotection induces isomerization); as a
result the deprotected linear tetrapeptide 23 was treated with CbzꢀCl in presence of AgCN. The
reaction proceeded with a change in the conformation to yield 29 (Scheme 14). The isolated
product was in cis conformation (confirmed from 2D NMR studies, SI).
The compound 29 on hydrolysis followed by EDC mediated peptide coupling with LꢀValꢀ
OMe.HCl proceeded with Cbz deprotection and conformation change to yield the pentapeptide
24 which was indistinguishable to the previously synthesized 24 (Scheme 11) in all aspects.
The pentapeptide 24 on reprotection exhibits similar trend as that of the tetrapeptide 23 on Cbz
protection 24 yield 30 (Scheme 14).
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BocHN
O
BocHN
O
CO Me
CO Me
2
2
O
O
O
O
Cbz-Cl, AgCN
HN
HN
NH
NH
o
C H , 60 C
N
6
6
N
93%
NH
NCbz
2
3
29
0
1
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8
9
0
1
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3
4
5
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7
8
9
0
CO Me
2
HN
BocHN
1
. LiOH, THF:H O
Cbz-Cl, AgCN
2
O
o
O
C H , 60 C
2
. L-Val-OMe.HCl, EDC
HOBT, DIPEA, DCM
6 6
O
O
90%
HN
NH
N
2
4
NH
CO Me
2
HN
BocHN
O
O
O
O
HN
NH
N
NCbz
30
Scheme 14. Effect of isomerization after Cbz reprotection of linear peptides.
In order to investigate the effect of reprotection of δ – nitrogen on cyclic peptide 18T with a
trans conformation was subjected to Cbz protection to yield 31 (Scheme 15).
O
O
AgCN, Cbz-Cl
NH
NH
HN
HN
O
O
o
O
O
C H , 60 C, 90%
6
6
NH
NH
HN
Pd / C, H
2
HN
N
NH
N
NCbz
O
O
MeOH, 4h
O
O
18T
31
Scheme 15. Cbz reprotection of 18T and deprotection of 31
NMR analysis shows an upfield shift of the αꢀhydrogen to 4.02 ppm from 4.72 indicating a
crossover to cis conformation (Figure 20).
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Figure 20. 1H NMR and HMQC of Cbzꢀprotected cisꢀcyclic peptide 31.
The compound 31 had a cis conformation, interestingly removal of the Cbz group from the δꢀN
under hydrogenolysis condition yielded the deprotected cyclic peptide 18T with a trans
conformation (Scheme 15).
NMR analysis of 18T and tallying it with that of previously synthesized 18T indicated that both
the compounds are indeed identical.
CONCLUSION
δꢀAzaproline, exclusively adopts a trans conformation in simple amides to tripeptides when its δꢀ
N is protected with a carbamate group and changes over to cis conformation during deprotection
which was confirmed by NMR spectroscopy and chymotrypsinꢀmediated digestion studies. In
longer linear and cyclic peptides due to an in situ deprotection the behavior reverses but the
reluctance of isomerization holds true in all the cases. This behavior of δꢀazaproline will be
beneficial in case of solid phase peptide synthesis where its deprotected from in longer peptides
will stabilize the trans conformation thereby minimizing aggregate formation. Theoretical
studies give a clear insight into the mechanism of isomerization during protection or
deprotection. The behavior of unprotected δꢀazaproline, in case of tripeptides remains the same
even if the δꢀN is protonated, which was evident from the NMR studies of 16 which despite
being an hydrobromide salt, adopted a cis conformation. Interesting observation is the chemical
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shift values of α ꢀ H of δꢀazaproline, that is upfield for cis ꢀ conformer than the trans ꢀ
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conformer, cis – trans conformers of proline also exhibit similar trend. During the course of our
work, a related work has been published by Jamart ꢀ Grégoire et. al. where they tried to prove
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similar preference of δꢀazaproline in simple tripeptides with the help of IR and NMR studies.
For the first time, δꢀazaproline has also been used for the synthesis of medicinally important
diketopiperazines, which are employing both the “N”s. Among several substituted prolines, δꢀ
azaproline is unique because of its ability to stabilize one conformer exclusively and the δ ꢀ N
provides a much required site for the spatioꢀtemporal control of bioactive peptides e.g.
prolyloligopeptidase inhibitors with a suitable protecting group or a photo labile group. Such
applications of δꢀazaproline with a photo labile protecting group in biological systems are
ongoing and will be reported shortly. Effects of non carbamate protecting groups on the cisꢀtrans
profile of δ – azaproline are also being investigated.
EXPERIMENTAL SECTION
General Methods.
All the reactions were done in argon atmosphere with dry solvents and reagents unless otherwise
mentioned. Reagents purchased from commercial sources were used directly except DCM, DMF
(
dried from CaH ), THF, benzene, toluene (dried over sodium). Column chromatography was
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done on a 230 – 400 mesh silica gel. Thinꢀlayer chromatography (TLC) was carried out on
aluminum sheets, Silica Gel 60 F254 (layer thickness 0.25 mm). Visualization of the developed
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chromatogram was performed by ceric ammonium molybdate (CAM) or KMnO stains. H and
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3
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ꢀD spectra were recorded at 300, 400 or 500 MHz and C NMR on 75, 100 or 125 MHz, using
CDCl , CD OD or D O as solvent. Chemical shifts (δ) are given in parts per million. The
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following abbreviations are used for multiplicity of NMR signals: s = singlet, d = doublet, t =
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triplet, m = multiplet, br = broad. High resolution mass spectra (HRMS) were measured in a
QTOF I (quadrupoleꢀhexapoleꢀTOF) mass spectrometer. Kinetics was measured by a diode
array spectrophotometer with a thermostated cuvette holder.
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1
ꢀBenzyl 3ꢀmethyl 2ꢀ(2ꢀ(((9Hꢀfluorenꢀ9ꢀyl) methoxy) carbonylamino)ꢀ3ꢀmethylbutanoyl)
pyrazolidineꢀ1, 3ꢀdicarboxylate, (4).
Fmoc protected LꢀValine (0.689 g, 2.03 mmol) was stirred with oxalyl chloride (0.72 ml, 8.4
mmol) in DCM at rt. for 4 h. The deep yellow (to red) solution was then freed from DCM and
excess oxalyl chloride in vacuo.
The acid chloride generated above was redissolved in dry benzene and added to a benzene
solution of 3 (0.447 g, 1.7 mmol). To the above mixture AgCN (0.453 g, 3.38 mmol) was added
o
and refluxed at 60 C for 30 min. On completion the reaction (TLC) benzene was removed, the
mixture was extracted with EtOAc, filtered through a sintered funnel. The organic layer was then
washed with satd. NaHCO followed by water and brine. Removal of the organic layer yielded
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the crude diasteriomeric mixture which was separated by a flash column chromatography (25%
ethyl acetate in petroleum ether). Overall yield 83% (0.826 g, 1.41 mmol, mixture 0.099 g).
(S)ꢀ1ꢀBenzylꢀ3ꢀmethylꢀ2ꢀ((R)ꢀ2ꢀ(((9Hꢀfluorenꢀ9ꢀyl)methoxy)carbonylamino)ꢀ3ꢀmethylbuꢀ
tanoyl)pyrazolidineꢀ1,3ꢀdicarboxylate, (4a).
1
Yield, 35%, 0.35 g, 0.6 mmol. H NMR (300 MHz, CDCl ) δ 7.73 (d,
3
O
MeO C
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N
2H, J = 7.5 Hz), 7.57 (d, 2H, J = 7.5 Hz), 7.24 – 7.39 (m, 9H), 5.59 (d,
NHFmoc
NCbz
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.23H, J = 9 Hz), 5.05 – 5.30 (m, 4H), 4.62 – 4.73 (m, 0.77H), 4.17 –
4.41 (m, 3H), 3.74 (s, 0.86H), 3.61 (s, 2.13H), 3.35 – 3.41 (m, 0.35H), 3.04 – 3.16 (m, 0.65H),
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2.12 – 2.38 (m, 4H), 0.82 – 1.02 (m, 6H). C NMR (75 MHz, CDCl ) δ 176.3, 170.8, 170.5,
3
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