Pyrrolidine ring puckering and prolyl amide bond configurations of 2-methyl-allo-hydroxyproline-based dipeptides

Article abstract of DOI:10.1039/c9ob00150f

An expeditious method for the synthesis of homo and heterochiral dipeptides containing l-alanine and d/l 2-methyl allo-hydroxyl prolines was developed using direct aminolysis of bicyclic lactones derived from d/l alanine. The impact of C-2 methylation and its spatial orientation on the pyrrolidine ring puckering and prolyl amide bond configuration was ascertained by solution NMR studies. The present studies reveal that C-2 methylation causes the prolyl amide bond to exist exclusively in the trans geometry in both homo- and heterochiral dipeptides. However, the spatial orientation of the C-2 methyl group and its i + 2 position in appropriately capped model dipeptides may nucleate into a turn like structure.

Full text of DOI:10.1039/c9ob00150f

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Pyrrolidine Ring Puckering and Prolyl Amide Bond Configurations
of 2‐methyl‐allo‐hydroxyproline‐based dipeptides†
Vinay Shankar Tiwari,a,c Gajendra Singh,b,c Gurudayal,b Ravi Sankar Ampapathi*b,c and Wahajul
Received 00th January 20xx,
Accepted 00th January 20xx
Haq*a,c
DOI: 10.1039/x0xx00000x
An expeditious method for the synthesis of homo and heterochiral dipeptides containing L‐alanine and D/L 2‐methyl allo‐
hydroxyl prolines were developed using direct aminolysis of bicyclic lactones derived from D/L alanine. The impact of C‐2
methylation and its spatial orientation on the pyrrolidine ring puckering and prolyl amide bond configuration was
ascertained by solution NMR studies. The present studies reveal that C‐2 methylation causes the prolyl amide bond to exist
exclusively in the transgeometryin both homo‐ and heterochiral dipeptides. However, the spatial orientation of C‐2 methyl
group and its i+2 position in appropriatly capped model dipeptides may nucleate into a turn like structure.
www.rsc.org/
group restricts both ϕ and preceding ω torsion angles to about
Introduction
‐60o and 180o respectively.11
Proline is considered to be unique among the other coded
natural amino acids because it induces helicity andβ‐turns in
peptides and proteins owing to its cyclic structure.1 The
pyrrolidine ring of proline exhibits at least in two distinct
puckered conformations namely C‐exo and C‐endo with
respect to the α‐carboxylic group.2 Several analogues of proline
are reported having asubstitution in the pyrrolidine ring. It has
been observed that puckering preferences of pyrrolidine ring
and its impact on peptide bond geometry by the introduction of
proline analogues in peptide sequence depends on the position
of these substituent and the nature of the substituting groups.3
In addition to the substitution in pyrrolidine ring, substitution of
α‐proton byanalkyl group is also known to significantly
influence the puckering phenomenon as well as amide bond
geometry.4
The hydroxyproline, another interesting naturally occurring
nonproteinogenic amino acid plays pivotal role in
a
conformation of collagen‐like peptides and its applications.12
The hydroxyl proline is formed by hydroxylation at the C‐4
position of pyrrolidine ring of proline as a post‐translational
modification, which provides an additional site for non‐covalent
interactions and thereby enhances the overall stability of
peptides and proteins. The overall stability of the collagen triple
helix is modulated by the content of hydroxyl proline residues
as well as the stereoelectronic effect of the C‐4 substituents.13
Conformational properties of both 4‐hydroxyproline and allo‐4‐
hydroxyproline have been well documented in the literature
and it was concluded that 4‐hydroxyproline favors C‐exo
pyrrolidine ring puckering and favours trans prolyl amide bond
configuration compared to proline. Conversely, allo‐4‐
Cα‐methylated analogs of coded amino acids have got
considerable interest in peptide research due to their enhanced
conformational rigidity and ability to induce helicity in the
polypeptide chain.5 The cyclic imino acids like proline and
hydroxyproline play a significant role in biological recognition
and signal transduction where the cis or trans form of the
peptide bond is very crucial.6 In the quest of design and
development of conformationally rigid peptides with target
selectivity, it is often essential for proline derivatives to exist in
a single conformation. Consequently, a number of proline
analogs with substitutions at C‐2,7 C‐3,8 C‐49 and C‐510 of
pyrrolidine ring has been developed and studied for their
conformational behaviour.7‐10 Among them, 2‐methylproline is
found to be the most constrained amino acid as the C‐2 methyl
hydroxyproline favors C‐endo pyrrolidine ring puckering and
cis prolyl amide bond conformation.14 However, conformational
preferences of quaternary proline and hydroxyl proline are
under explored owing to their challenging synthetic
methodology, and difficulties to obtain them in chirally pure
form. The synthesis of C‐2 methylated 4‐hydroxyproline is
reported in the literature starting from 4‐hydroxyproline using
different chiral auxiliaries and chiral reagents.15 However the
synthesis of the other diastereomer i.e. allo‐4‐hydroxy‐2‐
methylproline is challenging due to the fact that allo‐4‐
hydroxyproline is not found in nature and therefore is highly
expensive.16 Only one method for the synthesis of 2‐methyl‐
allo‐hydroxyproline is reported employing the chiral
epichlorhydrine15d but to the best of our knowledge, there is no
report describing the impact of C‐2 methylation on allo‐
hydroxyproline conformation.
a. Medicinal and Process Chemistry Division, CSIR‐Central Drug Research Institute,
Lucknow 226031, India.E‐mail: w_haq@cdri.res.in
b. NMR Centre, SAIF, CSIR‐Central Drug Research Institute, Lucknow 226031,
India.E‐mail: ravi_sa@cdri.res.in
c. Academy of Scientific and Innovative Research, New Delhi 11000.
†Electronic
Supplementary
Informaꢀon
(ESI)
available:See
DOI: 10.1039/x0xx00000x
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Journal Name
PO
PO
H
N
d
H
N
Reagents and conditions: a) L‐Ala‐NHMe, dry pyridine, 60o C, 6
h; b) (i) H2, Pd/C, Methanol, 1 h; (ii) Ac2O, rt, (iii)K2CO3,
Methanol, rt, 1 h.
N
H
O
N
N
c
O
Cbz
O
Cbz
5a (2R,4R & P = THP)
b (2S,4S & P = MOM)
HO
O
6a (2R,4R & P = THP)
b (2S,4S & P = MOM)
H
O
a
N
b
e
Scheme‐1Synthesis of dipeptides containing C‐2 methylated D
and L Allo‐hydroxyproline at (i+1) position.
N
N
f
O
Cbz
Cbz
HO
HO
1a (2R,4R)
b (2S,4S)
4a (2R,4R)
b (2S,4S)
H
H
N
N
N
H
N
H
O
N
O
N
Cbz
O
Cbz
O
Recently we have developed a diastereoselective method
for the synthesis of allo‐4‐hydroxy‐2‐methylproline starting
from alanine.17 This method provides easy and affordable
access to 2‐methyl‐allo‐hydroxyproline enantiomers, which
enable us to explore the conformational preferences of C‐2
methylated allo‐hydroxyproline containing peptides.
7b (2S,4S)
7a (2R,4R)
Reagents and conditions: a) MeNH2.HCl, NaHCO3,
toluene:water (8:2 v/v) 80ºC, 12 h; c) DHP, PPTA, 12 h or MOM‐
Cl, DIPEA, DMAP, DCM, 24 h; d) (i) H2, Pd/C, Methanol, 1 h; (ii)
Cbz‐L‐Ala‐OH, HBTU, TEA, ACN, 12 h; e) Acetyl chloride,
Methanol, 30 min; f) Dowex 50, Methanol, 60ºC, 5 h.
Earlier reports established that, C‐4 hydroxy group in allo‐4‐
hydroxyproline induces C‐endo pyrrolidine ring puckering and
cis prolyl amide bond conformation.14 Conversely C‐2
Scheme‐2 Synthesis of dipeptides containing C‐2 methylated D
and L allo‐hydroxyprolines at (i+2) position.
methylation of proline induces,
C‐exo pyrrolidine ring
puckering and trans prolyl amide bond conformation.18
Therefore, it would be rather intriguing to explore the
conformational preferences of proline derivatives, having
boththese substituent groups together in the same molecule,
as in the case of allo‐4‐hydroxy‐2‐methylproline.
hydroxyproline N‐methylamides.Therefore, we modified our
strategy and lactone 1a and 1b were subjected to aminolysis by
methylamine hydrochloride in
a toluene‐water biphasic
medium using sodium bicarbonate as a base at 80o C for
6htogive the corresponding N‐methyl amide 4a and 4b
(Scheme‐2). Cbz‐deprotection of compounds 4a and 4b was
achieved by hydrogenolysis and the resultant secondary amines
were coupled with Cbz‐L‐alanine using EDC.HCl and HOBt in
DMF, but we did not get the expected dipeptide instead an
inseparable mixture of products were seen on TLC. Changing
coupling reagents to IBCF/NMM and HBTU/DIEA also failed to
give the expected product. We speculated that the free
hydroxyl group at C‐4 creates problem in the coupling.
Therefore, we protected the C‐4 hydroxyl group of 4a with THP
and 4b with MOM to get compound 5a and 5b respectively.
Initially, when THP protection was used as in case of compound
5a, it gave an inseparable mixture of diastereomers and the
NMR data of the protected compound 5a, became further
complicated due to the existence of Cbz‐rotamers. Later on, we
switched to MOM protection of C‐4 hydroxy group as in case of
compound 5b for better clarity of data and to study the role of
the free C‐4 hydroxy group on peptide bond conformation. The
choice of the hydroxyl protecting group was made based on our
own experience with this sterically hindered amino acid.19 The
N‐Cbz‐protection of compound 5a and 5b was removed by
hydrogenolysis followed by coupling with Cbz‐L‐alanine and
HBTU as coupling reagent which afforded the dipeptides 6a and
6b. Finally, the THP‐protection of compound 6a was removed
using acetyl chloride in methanol to get compound 7a and the
MOM‐protection of compound 6b was removed by stirring with
Results and discussion
The conformational preferences of proline‐containing peptides
depend mainly on the position of the proline residue in the
peptide sequence. Proline residue if is placed either at (i+1) or
(i+2) positions, will induce a turn like structure. Therefore, two
sets of model peptides (2a, 2b, 3 and 7a, 7b) were synthesized
to study the conformational preferences of C‐2 methylated allo‐
hydroxyprolines at both these positions.
Synthesis of dipeptides (2a, 2b, and 3) with C‐2‐methyl‐allo‐
hydroxyproline at (i+1) position:
The starting bicyclic pyrrolidine lactones 1a and 1b were
synthesized starting from alanine as described in our previous
report.17 The direct aminolysis of bicyclic pyrrolidine lactone 1a
and 1b with L‐alanine N‐methylamide resulted in dipeptides
Cbz‐D‐(α‐me)allo‐Hyp‐L‐Ala‐NHMe2a and Cbz‐L‐(α‐me)allo‐
Hyp‐L‐Ala‐NHMe2b respectively at elevated temperature
(Scheme‐1). Compound 2b was subjected to hydrogenolysis
followed by N‐acetylation using acetic anhydride to give acetyl‐
L‐(α‐me)allo‐Hyp‐L‐Ala‐NHMe
3 along with a small amount of di‐
acetylated product which in turn was selectively hydrolyzed to
desired mono‐acetylated product
in methanol.
3 using potassium carbonate
Dowex 50X acidic resin in methanol to get compound 7b
.
Furthermore, to validate the impact of C‐2 methylation on
conformation of allo‐hydroxyproline, we synthesized
homochiral model dipeptides Boc‐L‐allo‐Hyp‐L‐Ala‐NMe 11 and
Cbz‐L‐Ala‐L‐allo‐Hyp‐NMe 13, starting with L‐hydroxyproline
Synthesis of dipeptides (7a, and 7b) with C‐2‐methy‐allo‐
hydroxyproline at (i+2) position:
Direct aminolysis of lactone 1a and 1b with methylamine failed
miserably to give the corresponding C‐2 methylated allo‐
(Scheme‐3).The L‐hydroxyproline
8 was first subjected to
esterification, then Nα Boc protection followed by stereo‐
inversion at C4 using PPh3/DEAD and PNBA under Mitsunobu
condition20 yielded the fully protected L‐allo‐hydroxyproline
diester
9. The diester 9 was hydrolyzed to give Boc‐L‐allo‐
hydroxyproline 10 which was either reacted with L‐alanine N‐
2 | J. Name., 2012, 00, 1‐3
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ProH
(pro‐S),

Ala‐H

in peptide 7a and other peptides,
suggesting that prolyl amide bond exists
Reagents and conditions: a) (i) SOCl2, MeOH, 0o C to rt, 3 h; (ii)
Boc2O,TEA, DCM, 0o C to rt, 2 h; (iii) PNBA, PPh3, DEAD, THF, rt,
18 h; b) 1N aq. NaOH, MeOH:H2O:THF (1:1:3 v/v), rt, 3h; c)
MeNH2.(2M in THF) or L‐Ala‐NMe, HBTU, TEA, ACN, rt, 12 h; d)
(i) HCl/dioxane, rt, 30 min; (ii) Cbz‐L‐Ala‐OH, HBTU, TEA, ACN, rt,
12 h.
Figure 1A
Scheme‐3. Synthesis of homochiral dipeptides containing L‐
allo‐hydroxyproline.
methyl amide to give the model dipeptide 11 or with
methylamine to give the corresponding Boc‐L‐allo‐
hydroxyproline N‐methyl amide 12
ofcompound 12 was removed and the corresponding amine
was coupled with Cbz‐L‐alanine to give the model dipeptide 13
. The N‐Boc group
.
Solution conformational studies were carried out by
employing NMR techniques on 5 ‐10 mM peptides, in DMSO‐d6.
A single set of resonances in the NMR spectra indicated the In
the NMR time scale, either
a predominantly single
conformation or averaging among several conformations21
might lead to an average structure and therefore we observe a
single set of resonances for the heterochiral dipeptides (2a and
7a) and homochiral dipeptides (2b, 3, 6b, 7b, 11, and 13).
Figure 1B
However, broadness was observed for the N‐Me resonances
which may be due to the quadrupolar interactions, which is a
quite common phenomenon among N‐methylated peptides.
Figure 1A: Chemical structure showing characteristic ROESY
correlations.1B)ROESY spectrum of 7a. The Characteristic rOes
ProH(pro‐S)ProH
Ala‐HProH(pro‐S), Pro‐OH
, N‐Me NH Ph‐CH2, N‐Me NH

, ProH(pro‐R)ProH
ProH(pro‐R), N‐Me NH
Pro‐OH are marked as 1‐8.
; Ala‐HProH(pro ;
‐R)
For compound 7a, characteristic medium range rOe was

 Ala‐
observed for ProHpro‐SProH
RProH was observed as illustrated in Figure 1a, indicating
that pyrrolidine ring exists in the C ‐endo conformation.
, and a weak rOe for ProHpro‐
H





However, when the stereocenter of C‐2 and C‐4 positions of
pyrrolidine was inverted as in compound 7b, interestingly
similar type of ring puckering was observed as evident from the
similar set of rOe interactions. These observations indicate that
changing the stereocenters at C‐2 and C‐4 position of
pyrrolidine ring has no significant effect on the pyrrolidine ring
puckering.
exclusively in trans configuration (Figure 1).The possibility of
intramolecular H‐bonding between C‐4 OH group of the C‐2
methyled allo‐hydroxyproline and its carbonyl group was ruled
out by the observed rOe pattern and VT NMR studies.(See SI)
To further support our finding we have also studied the
compound 11 and 13, by removing the methyl group at the C‐2
position of pyrrolidine ring.However, from the 1H NMR spectra
it is evident that these peptides were showing two distinct
populations, which were later identified astrans and cis
population in 1:0.25 ratio respectively.(See SI) These results
clearly indicate that the C‐2 methyl group locks the
configuration of the prolyl amide bond exclusively in the trans
form.
Variable temperature (VT) NMR studies carried out on 7a
,
from 303K to 343K shows, minimum deviation in the chemical
shifts (Δδ/ΔT = 1.60 ppb) in N‐Me NH as compared to Ala NH,
which show higher Δδ/ΔT (4.65 ppb) values, suggesting its
participation in H‐bonding. Aggregation study of peptide 7a
from 0.25 mM to 16 mM, was not showing any significant
changes in chemical shiftsof both the amide protons, indicating
that the nature of hydrogen bonding is intramolecular.
Furthermore, the significant rOes observed forProHpro‐SAla‐
Restrained Molecular Dynamics Simulation Study:
The restrained molecular dynamics (MD) of peptide 7a
‐7b, and
H, N‐MeNH Ala‐H, N‐Me NHPh‐CH2, N‐MeNHPro‐OH
further supports that N‐Me NH is participating in 10‐membered
hydrogen bonding with Cbz‐CO as shown in Figure1B and 2.We
2a
‐
2b were performed on the Discovery studio client
programme using CHARMm force field22 with default
parameters during the simulation. MD simulations were
performed on peptides using experimentally derived dihedral
angle restraints and distance restraints. The lowest energy
have also noticed the rOe of
ProH(pro‐R)Ala‐H,
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conformations derived from these simulations for the peptide discourages turn like structure while placed at the i+2 position
7a are presented in Figure 3, whereas for the other peptides in homo‐ and heterochiral dipeptides. However, in our studies,
we found that 2‐methylated allo‐hydroxy proline nucleate β‐
turn like structure only when it is present at the i+2 position and
that too only in the heterochiral dipeptides. Therefore, in this
study we have observed that the position of 2‐methyled allo‐
hydroxy proline in peptide chain, as well as the spatial
arrangement of C‐2 methyl group, plays a crucial role in the
formation of a turn like structure.
Conflicts of interest
please see the SI.
Figure: 2 A) Average structure of peptide 7a, showing 10‐
The authors declare no competing financial interest.
membered hydrogen bonding between N‐Me NH and Cbz CO.
B) Top 20 minimum energy structures were superimposed for
peptide 7a, where side chain protection group removed after
superimposition, for clarity.
Acknowledgements
V.S.T. thanks UGC New Delhi and G.S thanks to CSIR‐New Delhi for
financial support in form of fellowship. We also thank the SAIF
division, CSIR‐CDRI, for the analytical facilities. CDRI MS
No.125/2018/WH.
Notes and references
1
(a)M.Goodman, S. Ro, Burger’s medicinal chemistry and drug
discovery. In Peptidomimetics for Drug Design; M. E. Wolff,
Ed.; Wiley: New York,1995, pp 803; (b) S. K. Brahmachari, R. S.
Rapaka, R. S. Bhatnagar, V. S. Ananthanarayanan,
Biopoly.1982, 21, 1107.
(a) R.Deslauriers, I. C. Smith, R. Walter, J. Bio. Chem.1974, 249
7006; (b) Y. K. Kang, J. Phy. Chem. B2007, 111, 10550.
(a) K. Y. Huang, J. C. Horng, Biochemistry, 2015, 54, 6186; (b)
L. S. Sonntag, S. Schweizer, C.Ochsenfeld, H. Wennemers, J.
Am. Chem. Soc.2006, 128, 14697.
(a) Y. K. Kang, H. S. Park, New J. Chem.2014, 38, 2831; (b).A.
Flores‐Ortega, A. I. Jiménez, C. Cativiela, R. Nussinov, C.
Alemán, J.Casanovas, J. Org. Chem.2008,73 (9), 3418.
2
3
,
4
5
6
7
8
H. Vogt, S. Brase, Org. Biomol. Chem.2007,
5
, 406.
Figure: 3 Molecular dynamics single structure of peptides A) 2a
and 2b. B) 7a and 7b, showing Cγ‐endo ring puckering and trans
prolyl amide bond conformation, where side chain protection
group removed for the clarity.
A. A. Morgan, E. Rubenstein,PLoS ONE2013
8(1): e53785.
M. I. Calaza, C. Cativiela,. Eur. J. Org. Chem.2008, 3427.
(a) S. Guesne, S.Comesse, C. K. Puchot, Lett. Org. Chem.2006,
3
, 315;(b) C. Mothes, C. Caumes, A. Guez, H. Boullet, T.
Grendrineau, S. Darses, N. Delsuc, R. Moumne, B. Oswald,
O.Lequin, P. Karoyan, Mol. 2013, 18, 2307; (c) D. P. Affron, O.
A. Davis, J. A. Bull. Org. Lett.2014, 16, 4956. (c) R. Feng, B.
Wang, Y. Liu, Z. Liu, Y.Zhang, Eur. J. Org. Chem.2015, 142.
The superimposed structures for all peptides showing almost
similar Cγ‐endo pyrrolidine ring orientation. The single average
structure of a peptide with C‐endo conformation is also
9
(a) M. Tamaki, G. Han, V. J. Hruby, J. Org. Chem.2001, 66,
presented in Figure 3.
1038;(b) M. Tamaki, G. Han, V. J. Hruby, J. Org. Chem.2001,
66, 3593;(c) J. R. Del Valle, M. Goodman, Angew. Chem., Int.
Ed.2002, 41, 1600; (d) J. Y. Zhang, W. Wang, C. Y. Xiong, V. J.
Hruby, Tetra. Lett.2003, 44, 1413.
Conclusions
10 (a) E. A. A. Wallen, J. A. M. Christiaans, T. J. Saarinen, E. M.
Jarho, M. M. Forsberg, J. I. Venalainen, P. T. Mannisto,
J.Gynther, Bioorg. Med. Chem.2003, 11, 3611;(b) A. V. Moro,
E. R. T. Tiekink, J. Zukerman‐Schpector, D. S. Ludtke, C. R. D.
Correia, Eur. J. Org. Chem.2010, 3696; c) M. P. Brun, A. S.
Martin, C. Garbay, L. Bischoff, Tetra. Lett.2003, 44, 7011;(d) B.
M. Trost, E. J. Donckele, D. A. Thaisrivongs, M. Osipov, J. T.
Masters, J. Am. Chem. Soc.2015, 137, 2776; (e) V.J. Colandrea,
I. E. Legiec, P. Huo, L. Yan, J. J. Hale, S. G. Mills, J. Bergstrom,
D. Card, G. Chebret, R. Hajdu, C. A. Keohane, J. A. Milligan, M.
J. Rosenbach, G. J. Shei, S. M. Mandala, Bioorg. Med. Chem.
Lett.2006, 16, 2905;(f) R. Ali, G. Singh, S. Singh, R. S.
Ampapathi, W. Haq, Org. Lett.2016, 18, 2848.
In conclusion, we have thoroughly investigated the C‐2
methylated allo‐hydroxylproline containing peptides by
extensive solution NMR techniques. The study revealed that the
prolyl amid bond exclusively exists in the trans configuration in
both homo and heterochiral dipeptides and the pyrolidine ring
exist in Cγ‐endo ring puckering. Interestingly peptide 7a having
C‐2 methyl allo‐hydroxylproline at i+2 position, shows the the
participation of N‐Me NH proton in a 10‐ member H‐bonding
which is contradictory to earlier reports by De Poli et. al.11 In the
previous reports, it was concluded that 2‐Me proline nucleate
10‐membered β‐turn only when placed at the i+1 position and
4 | J. Name., 2012, 00, 1‐3
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11 M. De Poli, A. Moretto, M. Crisma, C. Peggion, F. Formaggio,
B. Kaptein, Q. B. Broxterman, C. Toniolo, Chem. Euro. J.2009,
15, 8015.
12 (a) T, Luo, K. L. Kiick, Euro. Poly. J., 2013,49, 2998; (b) T, Luo,
K. L. Kiick, Bioconj. Chem.,2017,28, 816.
13 R. S. Erdmann, H. Wennemers, J. Am. Chem. Soc.2012, 134
,
17117.
14 (a) R. J. Abraham, K. A. McLauchlan, Mol. Phy. Inter. J. Inter.
Bet Chem. Phy.; 1962, 5, 195; (b) M. D. Shoulders, F. W. Kotch,
A. Choudhary, I. A. Guzei, R. T. Rains, J. Am. Chem. Soc,
2010,132, 10857; (c) M. Kuemin, Y. A. Nagel, S. Scheizer, F. W.
Monnard, C. Ochenfeld, H. Wennemers, Angew. Chem. Int.
Ed. 2010, 49, 6324; (d) A. K. Pandey, D. Naduthambi, K. M.
Thomas, N. J. Zondlo, J. Am. Chem. Soc, 2013,135, 4333; (c) C.
V. Karunaratne,T. K. Weldeghiorghis, C. M. West, C. M. Taylor,
J. Am. Chem. Soc.2014, 136, 15170.
15 (a) T. Weber, D. Seebach, Helv. Chim. Acta1985, 68, 155; (b) S.
Nagumo, M. Mizukami, N. Akutsu, A.Nishida, N. Kawahara,
Tetra. Lett 1999,40, 3209; (c) T. Sato, S. Kawasaki, N. Oda, S.
Yai, S. A. A.El Bialy, J. Uenishi, M. Yamauchi, M. Ikeda, J. Chem.
Soc. Perk. T. 12001, 2623; (d) L. Kolaczkowski, D. M.Barnes,
Org. Lett.2007, 9, 3029.
16 (a) B. R. Huck, S. H. Gellman, J. Org. Chem.2005,70, 3353; (b)
D. J. Pippel, L. K. Young, M. A. Letavic, K. S. Ly, B. Naderi, A.
Soyode‐Johnson, E. M. Stocking, N. I. Carruthers, N. S. Mani, J.
Org. Chem.2010, 75, 4463.
17 V. S. Tiwari, R. Murugula, S. R. Yadav, W. Haq, Tetra.
Asy.,2016, 27, 865.
18 (a) N. G. Delaney, V.Madison, J. Am. Chem. Soc., 1982104
,
6635. (b) M. G. Hinds, J. H. D. Welsh, M. Brennand, J. Fisher,
M. J. Glennie, N. G. J. Richards, D. L. Turner, J. A.Robinson, J.
Med. Chem. 1991,34, 1777.
19 The C‐4 hydroxyl protection of compound 4b was attempted
either by TBDMS or by OtBu but both methods resulted in
poor yield (< 40%) mainly due to steric hindrance of the bulky
protecting groups and cis orientation of C2 amide group.
Therefore, relatively smaller C4‐hydroxyl protecting group like
MOM was used to get high yield (75%) of protected
compound 5b
.
20 M. B. Andrus, W. Li, R. F. Keyes, J. Org. Chem.1997, 62, 5542.
21 Neysa Nevins, Daniel Cicero, and James P. Snyder, J. Org.
Chem., 1999, 64, 3979.
22 A. D. Jr. MacKerell, D. Bashford, M. Bellott, R. L. Jr. Dunbrack,
J. D. Evanseck, M. J. Field, S. Fischer, J. Gao, H. Guo, S. Ha, D.
Joseph‐McCarthy, L. Kuchnir, K. Kuczera, F. T. K. Lau, C.
Mattos, S. Michnick, T. Ngo, D. T. Nguyen, B. Prodhom,W. E.
III Reiher, B. Roux,M. Schlenkrich,J. C. Smith,R. Stote, J. Straub,
M. Watanabe, J. Wio´rkiewicz‐Kuczera, D. Yin, and M. Karplus,
J. Phys.Chem. B. 1998, 102, 3586.
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Organic & Biomolecular Chemistry
Page 6 of 6
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DOI: 10.1039/C9OB00150F
Synthesis and conformational studies of C-2 methylated D and L-allo-hydroxyproline containing
pseudotetrapeptides is described.
1