Probing the folding induction ability of orthanilic acid in peptides: Some observations

Article abstract of DOI:10.1039/c3ra47039c

This paper describes the ability of orthanilic acid (2-aminobenzenesulfonic acid, ^SAnt) to promote folding when introduced in a peptide sequence. Three peptide sequences, containing orthanilic acid (^SAnt) with a sulfonamide moiety in the turn segment, have been synthesized in the solution phase using suitable coupling agents, and their structural aspects investigated using NMR and X-ray crystallographic studies. Solid- and solution-state conformational analyses reveal that the peptide sequences containing orthanilic acid in their backbone exist in a folded conformation featuring long-range 15-membered ring H-bonding. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c3ra47039c

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PAPER
Probing the folding induction ability of orthanilic
acid in peptides: some observations†
Cite this: RSC Adv., 2014, 4, 13018
Arup Roy,a Amol S. Kotmale,b Rupesh L. Gawade,c Vedavati G. Puranik,c
b
a
*
P. R. Rajamohanan and Gangadhar J. Sanjayan
This paper describes the ability of orthanilic acid (2-aminobenzenesulfonic acid, SAnt) to promote folding
when introduced in a peptide sequence. Three peptide sequences, containing orthanilic acid (SAnt) with
a sulfonamide moiety in the turn segment, have been synthesized in the solution phase using suitable
coupling agents, and their structural aspects investigated using NMR and X-ray crystallographic studies.
Solid- and solution-state conformational analyses reveal that the peptide sequences containing
orthanilic acid in their backbone exist in a folded conformation featuring long-range 15-membered ring
H-bonding.
Received 26th November 2013
Accepted 23rd January 2014
DOI: 10.1039/c3ra47039c
www.rsc.org/advances
sulfonamides and carboxamides have substantial conformational
disparity because of the presence of an additional H-bond acceptor.
Introduction
Moreover, the rotation barrier about the S–N bond is much lower as
compared to the C–N bond, which makes sulfonamides more
exible, leading them to remain in a twisted conformation which
may induce folding.7 Furthermore, the differences in the dihedral
angles of amides and sulfonamides are reason enough for them to
be used as a peptide bond replacement to generate intriguing
structures. Sulfonamides, as a result, have long been used as an
amide bond surrogate to develop various synthetic peptides.8
Unnatural amino acids are vital for understanding and mimicking
bio-constructs. In the recent past, chemists have taken advantage
of using unnatural amino acids to develop peptidomimetic-based
leads for drug discovery.1 This is primarily because of the proteo-
lytic stability of the unnatural amino acids as compared to their
natural counterparts, which makes them unsuitable to be used in
therapeutics. Moreover, the incorporation of unnatural amino
acids in a peptide sequence offers the possibility of generating
diverse secondary structures. Over the years, various research
groups have developed and utilized unnatural amino acids to
generate numerous peptidomimetic and foldamer molecules with
interesting secondary structural preferences (Fig. 1).2–4
Of the amino acids shown in Fig. 1, orthanilic acid deserves a
special mention, because SAnt can introduce a sulfonamide bond
into a poly-peptide chain. A sulfonamide bond possesses higher
proteolytic stability, polar character, and a tetrahedral sulfur atom
mimicking the intermediate formed during proteolysis. Sulfon-
amides therefore make up an important class of drugs, with many
pharmacological agents showing antibacterial, antitumor, anti-
carbonic anhydrase, diuretic, hypoglycemic, antithyroid, or
protease inhibitory activity,5 and many more.6 Although similar,
Result and disscusion
We have previously demonstrated the geometrical and
H-bonding similarity between sulfonamide and carboxamide by
aDivision of Organic Chemistry, CSIR-National Chemical Laboratory, Dr. Homi
Bhabha Road, Pune 411 008, India. E-mail: gj.sanjayan@ncl.res.in; Fax: +91-020-
2590-2629; Tel: +91-020-2590-2082
bCentral NMR Facility, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road,
Pune 411 008, India
cCentre for Material Characterizations, CSIR-National Chemical Laboratory, Dr. Homi
Bhabha Road, Pune 411 008, India
† Electronic supplementary information (ESI) available: 1H, 13C, DEPT-135 NMR,
2D study spectra, ESI mass spectra and theoretical study of new compounds are
included. CCDC [927811]. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/c3ra47039c
Fig. 1 Some of the important unnatural amino acids which have
frequently been used for generating various peptidomimetic
molecules.
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inserting orthanilic acid (SAnt) in place of anthranilic acid (Ant)
in the Ant–Pro (anthranilic acid–proline) turn segment which
showed the existence of strong 9-membered ring H-bonding.9
The results obtained were quite intriguing given the fact that
these groups have relatively different H-bonding and geomet-
rical preferences, which is well established.10 However, the
literature precedents and the geometric disparity of sulfon-
amides as compared to carboxamides are too signicant to be
content with a single result. Consequently, to explore further in
the direction of developing novel peptidomimetics and under-
standing their conformational preferences, we designed a range
of short chain oligopeptides featuring SAnt–Pro as the potential
turn inducer (Fig. 2).
Synthesis
The synthesis of the foldamer sequence bearing orthanilic acid
(sAnt) started with dimer 1 (Scheme 1), which on catalytic
hydrogenation furnished amine
2 in quantitative yield.
Scheme 1 Reagents and conditions: (i) H2, Pd–C, MeOH, 12 h; (ii) 2-
bromo-2-methylpropanoyl bromide, Et3N, DCM, 12 h, 90%; (iii) NaN3,
DMF, 75 ꢀC, 12 h, 81%; (iv) H2, Pd–C, 60 psi, 10 h; (v) Boc–Leu–OH,
EDC, HOBt, DCM, 10 h, 85%; (vi) LiOH, MeOH, H2O, 4 h; (vii) EDC,
HOBt, DCM, 10 h, 75%; (viii) sat. methanolic MeNH2, 8 h, 80%.
Coupling of amine 2 with 2-bromo-2-methylpropanoyl bromide
gave 3 in 90% yield, which was subjected to nucleophilic
substitution to furnish azide 4 in very good yield. The trimer
azide 4 was then reduced in the presence of H2, Pd–C and
coupled with Boc–Leu–OH in the presence of EDC$HCl as the
coupling agent to obtain the tetramer 6a in 85% yield. Ester
hydrolysis of 6a was followed by coupling with the free amine of
dipeptide 7,11 furnishing hexamer 8 in 75% yield. Hexamer 8 on
treatment with saturated methanolic MeNH2 produced the
methyl amide analog 9 in 80% yield. The results obtained
during the conformational analyses (vide infra) of oligomer 9
suggested that the Leu1 residue at the C-terminus had frayed,
presumably owing to the inherent exible nature of leucine.
This prompted us to replace the exible Leu residue at the
N-terminus with the conformationally rigid Aib and Pro,
developing two more sequences.
with Piv–Pro–OH, furnishing the tetramer 13a in 86% yield. Free
acid 13b, obtained aer the basic hydrolysis of tetramer 13a, was
coupled with the free amine of dipeptide 7 to get the hexamer 14 in
73% yield, which on reaction with sat. methanolic MeNH2 resulted
in the formation of the methyl amide analog 15 in 86% yield.
Solid-state conformational analysis
Extensive efforts to crystallize the oligomers resulted in the
formation of crystals of 9 (Fig. 3).12 Careful analysis of the crystal
To prepare the sequence with a-aminoisobutyric acid (Aib) at
the N-terminus, we started with the trimer amine 5 (Scheme 2)
which was coupled with Boc–Aib–OH, in the presence of
EDC$HCl as the coupling agent to obtain the tetramer 10a in 85%
yield. Ester hydrolysis of 10a was followed by coupling the acid
with the free amine of dipeptide 7, producing hexamer 11 in 82%
yield. Hexapeptide 11, containing the constrained amino acid Aib
at the N-terminus, upon treatment with sat. methanolic MeNH2
furnished the methyl amide analog 12 in 82% yield.
In order to access another sequence and obtain 15 bearing
proline at the N-terminus, free amine 5 (Scheme 2) was rst coupled
Scheme 2 Reagents and conditions: (i) Boc–Aib–OH, EDC, HOBt,
DCM, 10 h, 85%; (ii) LiOH, MeOH, H2O, 4 h; (iii) 7, EDC, HOBt, DCM, 10
h, 82%; (iv) sat. methanolic MeNH2, 8 h, 82%; (v) Piv–Pro–OH, EDC,
Fig. 2 Design strategy to synthesize oligomer sequences 9, 12 and 15 HOBt, DCM, 10 h, 86%; (vi) LiOH, MeOH, H2O, 4 h; (vii) 7, EDC, HOBt,
using orthanilic acid as a connecting entity to facilitate folding.
DCM, 10 h, 73%; (viii) sat. methanolic MeNH2, 8 h, 86%.
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data revealed the presence of three inter-residue H-bonding loca-
All of the oligomers were readily soluble in nonpolar organic
tions, and a 6-membered intra-residue H-bonded ring between NH solvents ([100 mM in CDCl3) at ambient temperature. Signal
˚
and S]O of the orthanilic acid moiety [d(C]O/H–N) ¼ 1.98 A, assignments were made unambiguously using a combination of
bond angle (N–H/O) ¼ 141ꢀ]. Of the three different types of inter- two-dimensional COSY, TOCSY, HSQC, HMBC and NOESY
residue H-bonding patterns, (i) one was a 10-membered ring (C10 experiments. Oligomers 9 and 12 exhibited sharp signals for
1
H-bonding) between the C-terminus amide NH and the carbonyl of Boc–CH3 in H NMR, whereas the oligomer 15 with proline at
˚
the proline moiety [d(C]O/H–N) ¼ 2.24 A, bond angle (N–H/O) the N-terminus showed multiple signals, suggesting the pres-
¼ 130ꢀ], (ii) one was a 7-membered ring between S]O of SAnt and ence of multiple conformers.
˚
N–H of Leu2 [d(C]O/H–N) ¼ 2.71 A, bond angle (N–H/O) ¼
The characteristic nOes supporting the folded conformation
123ꢀ], and (iii) one was an unusual, long range (15-membered) H- of 9 found in the solid-state were the inter-residue coupling
bonding ring between N–H of Aib1 and C]O Leu2 [d(C]O/H–N) between Aib1–CH3 (C10H) and NH5 along with Aib1–CH3
ꢀ
˚
¼ 2.02 A, bond angle (N–H/O) ¼ 167 ] (Fig. 3). The dihedral angle (C10H) and C33H (ESI, Fig. 6, S39†). Other important nOes were
values shed more light on the observed conformation of the olig- found between Pro–aH (C18H) and SAnt (C14H). Dipolar
omer 9 containing the sulfonamide connecting entity. As evident coupling was also observed between Pro–aH (C18H) with NH3
S
from the crystal structure, the torsion angles of Ant were: f ¼ and NH4. However, there were no nOes observed between Boc–
174.68ꢀ, q ¼ 5.01ꢀ, j ¼ 92.55ꢀ and u ¼ ꢁ55.31ꢀ. In the case of CH3 and the C-terminus methyl (C33H) as anticipated from the
proline, f and j were found to be ꢁ107.85ꢀ and 19ꢀ, respectively. single crystal X-ray diffraction analysis.
Thus, the solid-state conformational analysis clearly indicates that
For the oligomer 12, nOes that supported the folded
the torsional constraint of the orthanilic acid (SAnt) residue is a key conformation were between Pro–aH (C16H) and NH3, NH3 and
factor for the folded conformation seen in oligomer 9.
NH4, and C33H and Aib2–CH3 (C8H). Inter-residue nOes were
also observed between NH3 and NH2 (ESI, Fig. 11, S44†).
In the case of oligomer 15, inter-residue dipolar couplings were
observed between Pro2–aH (C17H) and NH2, Pro2–aH (C17H) and
Aib1–CH3 (C9H), and C-terminus methyl (C33H) and Aib1–CH3
(C9H). Also observed were nOes of Pro1–aH (C2H) with NH2 and
Aib2–CH3 (C30H) (ESI, Fig. 16, S49†). Similar nOe patterns of the
identical central residues in all the oligomers suggested the
prevalence of the folded conformation as anticipated.
Conformational investigation in solution-state
We undertook extensive NMR studies to provide insights into
the solution-state conformation of the oligomers. Details of the
peak assignments with spectra are provided in the ESI.†
To investigate the existence of intramolecular hydrogen-
bonding in the oligomers, we also undertook DMSO-d6 titra-
tions and variable temperature experiments (ESI, S31–S34†). All
of the NHs of 9 involved in intramolecular hydrogen bonding
showed very little shi in the titration studies [Dd (NH) < 0.25
ppm]. Only NH5 underwent a relatively larger chemical shi
change [Dd (NH5) < 0.7 ppm] on the incremental addition of
DMSO-d6, suggesting its non-participation in H-bonding.
Similar was the case for oligomer 12 where a considerable
change in chemical shi was observed for NH5 and NH1 [Dd
(NH) < 0.52 ppm], while the rest of the NHs showed a negligible
shi [Dd (NH) < 0.18 ppm]. Oligomer 15, on the other hand,
showed very little change in the chemical shi values for all of
the NHs [Dd (NH) < 0.25 ppm], except for NH3 [Dd (NH) < 0.49
ppm], implying the weakness of the H-bonding.
Variable temperature experiments of 9, 12 and 15 were
mostly in agreement with the observations from the other
studies, showing temperature coefficients (Dd/DT) > ꢁ3.8 ppb
Kꢁ1 for all NHs, except for NH2 of compound 9 and NH4 of 12
[(Dd/DT) for NH2 of 9 ¼ ꢁ6.5 ppb Kꢁ1 and (Dd/DT) for NH4 of 12
¼ ꢁ5.6 ppb Kꢁ1] (ESI, Fig. 1, S34†). This suggests that the 15-
membered H-bonding in compound 9 is relatively weak, espe-
cially at higher temperature. The same is applicable for the NH4
of compound 12.
All of the interesting results found during the solid- and
solution-state studies amplied our desire to investigate the
conformational features of the oligomers 12 and 15 which could
not be crystallized, in spite of the effort put in. This impelled us
to undertake nOe-based molecular modeling studies.
Fig. 3 Crystal (a) and molecular structure (b) of oligomer 9, and
different types of H-bonding observed in the solid-state (c–f).
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Solution state structural investigations
Experimental procedures
(S)-Methyl-1-(2-nitrophenylsulfonyl)pyrrolidine-2-carboxylate 1
NMR-based structures of compound 12 and 15 were derived
from nOe cross peaks by using the Maestro v9.3.518 program
L-Proline methyl ester hydrochloride (0.82 g, 4.96 mmol) was
added to a solution of 2-nitrobenzenesulfonylchloride (1.0 g,
4.51 mmol) in anhy. DCM (10 mL) at 0 ꢀC followed by the
addition of Et3N (1.45 mL, 10.38 mmol). The resulting mixture
was then allowed to attain room temperature and was stirred for
12 h. It was then sequentially washed with sat. NaHCO3, water,
dil. HCl and brine. The organic layer was then dried over anhy.
Na2SO4 and evaporated under reduced pressure to obtain the
crude product which on purication by column chromatog-
raphy furnished 1 as an off-white solid. Yield: 0.81 g (57%);
m.p.: 85–86 ꢀC; [a]D26: ꢁ100.0ꢀ (c ¼ 1.6, CHCl3); IR (CHCl3) n
(cmꢁ1) 3621, 3418, 3020, 1746, 1640, 1546, 1371, 1216, 1163,
¨
from Schrodinger (for a complete table, please refer to the ESI†).
The twenty lowest-energy superimposed structures of
compounds 12 and 15 showed root mean square deviations
˚
˚
(RMSDs) of 0.26 ꢂ 0.06 A and 0.15 ꢂ 0.06 A, respectively (Fig. 4).
The dynamic ensembled structures of compound 12 revealed
that the N-terminus did not reverse as expected when Aib was
put in place of Leu. The H-bonding pattern remained the same,
as observed for oligomer 9. However, the solution-state struc-
tural analysis of compound 15 showed the presence of a second
10-membered ring (C10 H-bonding) between the NH of SAnt
and the carbonyl of the pivaloyl moiety. Also, the 7-membered
ring H-bonding between S]O of SAnt and N–H of Leu2 seen in
the previous cases was no longer intact, which was obvious from
the DMSO titration studies. The rest of the H-bonding patterns
remained the same. Thus, the nOe-based structural investiga-
tions concluded that the oligomers display a folded structure.
1
770; H NMR (500 MHz, CDCl3) d: 8.08 (s, 1H), 7.71–7.69 (m,
2H), 7.63–7.62 (s, 1H), 4.58–4.57 (d, J ¼ 8 Hz, 1H), 3.66 (s, 3H),
3.62–3.60 (m, 1H), 3.55–3.52 (m, 1H), 2.29–2.22 (m, 1H), 2.10–
1.94 (m, 3H); 13C NMR (125 MHz, CDCl3) d: 172.2, 148.0, 133.6,
133.5, 132.7, 132.6, 131.6, 130.9, 124.0, 60.8, 52.3, 48.4, 30.8,
24.4; ESI MS: 337.04 (M + Na)+; anal. calcd for C12H14N2O6S: C,
45.85; H, 4.49; N, 8.91; found: C, 45.32; H, 4.73; N, 9.28.
Conclusion
In summary, oligomers carrying SAnt as the turn inducer
showed compact structures with a folded conformation, which
supports the ability of sulfonamides to promote folding.
Moreover, all the oligomers containing SAnt showed long-range
15-membered ring H-bonding involving four amino acid resi-
dues. These results further validate our observations that
(S)-Methyl-1-(2-aminophenylsulfonyl)pyrrolidine-2-
carboxylate 2
10% Pd–C (0.015 g) was added to a solution of 1 (0.15 g, 0.47
mmol) in methanol (6 mL). The reaction mixture was then
stirred at 60 psi under a hydrogen atmosphere for 8 h, followed
by ltration of the catalyst through celite, and the ltrate was
evaporated to obtain product 2 which was carried forward
without further purication.
orthanilic acid (2-aminobenzenesulfonic acid, SAnt) is
a
strong reverse-turn inducer when incorporated into peptide
sequences.13
(S)-Methyl-1-(2-(2-bromo-2-methylpropanamido)-
phenylsulfonyl)pyrrolidine-2-carboxylate 3
To a solution of 2 (4.0 g, 14.1 mmol) in dry DCM (25 mL), anhy.
ꢀ
Et3N (2.55 mL, 18.3 mmol) was added at 0 C followed by the
slow addition of 2-bromo-2-methylpropanoyl bromide (1.92 mL,
15.5 mmol). The resulting mixture was then allowed to attain
room temperature and was stirred for 12 h, following which it
was sequentially washed with sat. NaHCO3, water and brine.
The organic layer was then dried over anhydrous Na2SO4 and
evaporated under reduced pressure to obtain the crude product
which on purication by column chromatography furnished 3
as a viscous liquid. Yield: 5.5 g (90%); [a]2D6: ꢁ63.08ꢀ (c ¼ 1.3,
CHCl3); IR (CHCl3) n (cmꢁ1) 3349, 3020, 1740, 1688, 1588, 1338,
1215, 1154, 759; 1H NMR (400 MHz, CDCl3) d: 10.41 (s, 1H),
8.55–8.53 (d, J ¼ 8 Hz, 1H), 7.91–7.89 (dd, J ¼ 8.0, 1.6 Hz, 1H)
7.61–7.57 (t, J ¼ 7 Hz, 1H), 7.26–7.22 (t, J ¼ 7 Hz, 1H), 4.39–4.36
(dd, J ¼ 8.0, 2.5 Hz, 1H), 3.64–3.59 (m, 4H), 3.49–3.43 (m, 1H),
2.13–2.09 (m, 1H), 2.06 (s, 6H), 2.04–1.98 (m, 2H), 1.93–1.84 (m,
1H); 13C NMR (100 MHz, CDCl3) d: 171.9, 170.4, 136.5, 134.2,
129.6, 125.8, 124.0, 122.1, 60.4, 59.9, 52.5, 48.4, 31.7, 31.0, 24.5;
ESI MS: 455.01 (M + Na)+; anal. calcd. for C16H21BrN2O5S: C,
44.35; H, 4.88; N, 6.46; found: C, 44.62; H, 5.26; N, 6.08.
Fig. 4 Molecular and nOe-derived structures of compounds 12 (a)
and 15 (b) showing a folded conformation.
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Methyl((2-(2-azido-2-methylpropanamido)phenyl)sulfonyl)-L-
prolinate 4
Methyl((2-(2-(2-((tert-butoxycarbonyl)amino)-2-
methylpropanamido)-2-methylpropanamido)phenyl)-
sulfonyl)-L-prolinate 10a
Sodium azide (0.68 g, 10.5 mmol) was added to a solution of 3
(1.2 g, 3.5 mmol) in anhy. DMF (10 mL) and the reaction Tetramer 10a was obtained as a white uffy solid. Yield: 90ꢁ%1;
25
ꢀ
mixture was maintained at 70 ꢀC for 12 h. It was then m.p.: 67–69 C; [a]D : ꢁ12ꢀ (c ¼ 1, CHCl3); IR (CHCl3) n (cm
)
cooled to room temperature, following which EtOAc (40 mL) 3337, 3019, 2400, 1735, 1699, 1523, 1338, 1215, 1045, 928, 758,
was added and the organic layer was washed with water 669; H NMR (400 MHz, CDCl3) d: 10.04 (s, 1H), 8.58–8.56 (d,
1
and brine, dried over anhydrous Na2SO4 and evaporated 1H, J ¼ 8.54 Hz), 7.82–7.79 (d, 1H, J ¼ 8.03 Hz), 7.56–7.52 (m,
under reduced pressure to obtain the crude product 1H), 7.27 (bs, 1H), 7.19–7.15 (m, 1H), 5.03 (s, 1H), 4.33–4.30 (m,
which on purication by column chromatography gave 4 as 1H), 3.64 (s, 3H), 3.53–3.48 (m, 1H), 3.35–3.29 (m, 1H), 2.11–1.92
a viscous liquid. Yield: 0.88 g (81%); [a]2D6: ꢁ50.53ꢀ (c ¼ 0.95, (m, 3H), 1.86–1.77 (m, 1H), 1.61 (s, 6H), 1.49 (s, 6H), 1.43 (s, 9H);
CHCl3); IR (CHCl3) n (cmꢁ1) 3436, 3020, 2120, 1739, 1685, 13C NMR (100 MHz, CDCl3) d: 174.5, 173.2, 172.0, 155.0, 137.1,
1585, 1340, 1217, 1140, 770; 1H NMR (400 MHz, CDCl3) d: 134.2, 129.2, 124.8, 123.4, 122.3, 60.3, 57.4, 56.9, 52.4, 48.3, 30.8,
10.39 (s, 1H), 8.52–8.50 (d, J ¼ 8 Hz, 1H), 7.89–7.87 (dd, J ¼ 28.2, 25.3, 25.2, 24.9, 24.4; LC-MS: 477.15 (M + Na)+, 493.14 (M +
8.0, 1.5 Hz, 1H), 7.58–7.54 (t, J ¼ 7 Hz, 1H), 7.24–7.20 (t, J ¼ K)+; anal. calcd. for C25H38N4O8S: C, 54.14; H, 6.91; N, 10.10;
7 Hz, 1H), 4.39–4.36 (dd, J ¼ 8.0, 2.8 Hz, 1H), 3.60–3.54 (m, found: C, 54.43; H, 6.68; N, 10.28.
4H), 3.46–3.40 (m, 1H), 2.15–2.07 (m, 1H), 2.05–1.95 (m, 1H),
1.93–1.84 (m, 1H), 1.62 (s, 6H); 13C NMR (100 MHz, CDCl3) d:
Methyl((2-(2-methyl-2-((R)-1-pivaloylpyrrolidine-2-
171.9, 171.1, 136.1, 134.1, 129.7, 126.2, 124.0, 122.2, 64.6,
60.2, 52.4, 48.3, 31.0, 24.5; ESI MS: 418.07 (M + Na)+; anal.
calcd for C16H21N5O5S: C, 48.60; H, 5.35; N, 17.71; found: C,
48.29.; H, 5.11.; N, 17.09.
carboxamide)propanamido) phenyl)sulfonyl)-L-prolinate 13a
Compound 13a was obtained as a white uffy solid. Yield: 88ꢁ%1;
25
ꢀ
m.p.: 60–62 C; [a]D : ꢁ88ꢀ (c ¼ 1, CHCl3); IR (CHCl3) n (cm
)
3336, 3019, 2400, 1739, 1699, 1585, 1522, 1338, 1215, 1152, 762,
669; 1H NMR (400 MHz, CDCl3) d: 10.08rotamer (0.2H),
10.04rotamer (0.8H), 8.57rotamer (0.2H), 8.56–8.54rotamer (0.8H),
7.78–7.77 (m, 1H), 7.55rotamer (0.2H), 7.52rotamer (0.8H), 7.50–
7.49 (m, 1H), 7.15–7.12 (m, 1H), 4.66rotamer (0.2H), 4.64–
Methyl((2-(2-amino-2-methylpropanamido)phenyl)sulfonyl)-L-
prolinate 5
The crude product 5 was obtained from 4, following the
procedure mentioned for 2, and it was used for the next step
without further purication.
4.62rotamer (0.8H), 4.30–4.28
(0.8H), 4.26–4.25
rotamer
rotamer
(0.2H), 3.67–3.65 (m, 2H), 3.61rotamer (2H), 3.61rotamer (1H), 3.50–
3.46 (m, 1H), 3.32–3.27 (m, 1H), 2.25–2.22 (m, 1H), 2.04–2.00
(m, 2H), 1.98–1.91 (m, 2H), 1.87–1.77 (m, 3H), 1.55–1.54 (m,
6H), 1.23rotamer (7H), 1.23rotamer (2H).; 13C NMR (100 MHz,
CDCl3) d: 178.0, 173.1, 173.0, 171.9, 171.7, 171.6, 137.2, 134.1,
129.2, 129.1, 124.9, 124.8, 123.3, 123.2, 122.2, 122.0, 61.8, 61.6,
60.3, 60.1, 57.4, 52.3, 48.2, 39.1, 30.7, 27.4, 25.8, 25.6, 24.5. 24.4,
23.9; LC MS: 573.23 (M + Na)+, 589.20 (M + K)+; anal. calcd. for
Methyl-((2-(2-(2-((tert-butoxycarbonyl)amino)-4-
methylpentanamido)-2-methylpropanamido)phenyl)-
sulfonyl)-L-prolinate 6a
Representative procedure. EDC$HCl (0.142 g, 0.69 mmol)
was added to a solution of 5 (0.17 g, 0.46 mmol) and Boc–Leu–
OH (0.12 g, 0.50 mmol) in anhy. DCM at 0 ꢀC, followed by HOBt
(0.062 g, 0.46 mmol). The resulting mixture was then stirred at
C
25H36N4O7S: C, 55.95; H, 6.76; N, 10.44; found: C, 55.52; H,
6.99; N, 10.50.
0
ꢀC for 10 min and at room temperature for 12 h. To the
reaction mixture, 30 mL DCM was added and the organic layer
was washed sequentially with sat. NaHCO3, water, sat. KHSO4
and brine. It was concentrated under reduced pressure and
nally puried by column chromatography to furnish a white
solid. Yield: 0.24 g (85%); m.p.: 90–92 ꢀC; [a]D25: ꢁ82ꢀ (c ¼ 1,
CHCl3); IR (CHCl3) n (cmꢁ1) 3337, 3020, 2400, 1700, 1503, 1337,
((2-(2-(2-((tert-Butoxycarbonyl)amino)-4-methylpentanamido)-
2-methylpropanamido) phenyl)sulfonyl)-L-proline 6b
Representative procedure. LiOH$H2O (0.06 g, 1.3 mmol) was
added to a solution of 8 (0.2 g, 0.34 mmol) in methanol (5 mL),
followed by water (1 mL) at 0 ꢀC. Aer complete consumption of
the starting material (4 h), the solvent was evaporated under
reduced pressure, and the free acid was generated by treating
with sat. NaHSO4 solution followed by extraction with DCM (2 ꢃ
10 mL). Compound 6b obtained aer evaporation of the solvent
under vacuum was carried forward without further purication.
1
1215, 1155, 1022, 759, 699; H NMR (400 MHz, CDCl3) d: 10.12
(s, 1H), 8.63–8.61 (d, 1H, J ¼ 8.53 Hz), 7.82–7.80 (d, 1H, J ¼ 8.03
Hz), 7.58–7.54 (m, 1H), 7.20–7.16 (m, 1H), 6.93 (bs, 1H), 5.01 (bs,
1H), 4.34–4.31 (m, 1H), 4.19 (bs, 1H), 3.68 (s, 3H), 3.52–3.50 (m,
1H), 3.32–3.26 (m, 1H), 2.08–1.98 (m, 3H), 1.82–1.66 (m, 3H),
1.66 (s, 3H), 1.59 (s, 3H), 1.54–1.48 (m, 1H), 1.45 (s, 9H), 0.94–
0.91 (m, 6H); 13C NMR (100 MHz, CDCl3) d: 174.6, 172.9, 171.5,
137.3, 134.8, 129.8, 123.4, 123.1, 122.7, 79.4, 61.8, 57.4, 56.1,
53.4, 52.4, 49.7, 40.0, 30.9, 28.2, 26.0, 25.0, 24.9, 24.7, 24.6, 24.3,
24.1, 22.9, 22.7, 22.0; LC-MS: 605.25 (M + Na)+; anal. calcd. for
Methyl-2-(2-((2S)-1-((2-(2-(2-((tert-butoxycarbonyl)amino)-4-
methylpentanamido)-2-methylpropanamido)phenyl)sulfonyl)
pyrrolidine-2-carboxamido)-4-methylpentanamido)-2-
methylpropanoate 8
C
27H42N4O8S: C, 55.65; H, 7.27; N, 9.62; found: C, 55.81; H,
Representative procedure. A solution of free acid 6b (0.2 g
0.35 mmol) and dimer amine 12 (0.09 g, 0.38 mmol) in anhy.
7.05.; N, 9.75.
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DCM was cooled to 0 ꢀC. To this mixture, EDC$HCl (0.11 g, 0.52 8.43–8.41rotamer (0.2H), 7.79–7.77 (d, 1H, J ¼ 7.94 Hz), 7.60–7.56
mmol) was added followed by HOBt (0.05 g, 0.35 mmol), and (m, 2H), 7.22–7.19 (m, 1H), 7.06 (bs, 1H), 6.98–6.96rotamer (0.8H),
was stirred at 0 ꢀC for 10 min followed by 12 h at room 6.94rotamer (0.2H), 4.66–4.65 (m, 1H), 4.49–4.46 (m, 1H), 4.18–
temperature. DCM (30 mL) was then added to the reaction 4.17rotamer (0.2H), 4.15–4.12rotamer (0.8H), 3.68 (s, 3H), 3.65–3.61
mixture and the organic layer was washed sequentially with sat. (m, 2H), 3.15–3.10 (m, 1H), 2.20–2.18 (m, 1H), 2.07–2.01 (m,
NaHCO3, water, sat. KHSO4 and brine, concentrated under 2H), 1.91–1.76 (m, 5H), 1.70–1.68 (m, 1H), 1.55 (s, 3H), 1.54 (s,
reduced pressure and nally puried by column chromatog- 3H), 1.53–1.50 (m, 1H), 1.48 (s, 3H), 1.45 (s, 3H), 1.22rotamer (7H),
raphy to furnish a white solid. Yield: 0.2 g (75%); m.p.: 115–117 1.19rotamer (2H), 0.93–0.92 (m, 3H), 0.89–0.88 (m, 3H); 13C NMR
ꢀC; [a]2D5: ꢁ101.69ꢀ (c ¼ 1.18, CHCl3); IR (CHCl3) n (cmꢁ1) 3394, (100 MHz, CDCl3) d: 178.1, 174.5, 173.3, 173.2, 172.0, 171.9,
3019, 2400, 1674, 1523, 1338, 1215, 1046, 928, 755, 669; 1H NMR 171.1, 171.0, 170.8, 137.5, 134.8, 134.7, 129.6, 124.0, 123.9,
(400 MHz, CDCl3) d: 10.17 (s, 1H), 8.52–8.51 (d, 1H, J ¼ 7.93 Hz), 123.8, 123.2, 122.8, 62.3, 62.2, 61.7, 61.6, 57.4, 56.1, 52.3, 52.2,
7.83–7.82 (d, 1H, J ¼ 7.93 Hz), 7.61–7.58 (m, 1H), 7.43 (bs, 1H), 51.6, 49.4, 48.4, 48.3, 40.3, 40.2, 39.0, 30.8, 30.7, 27.4, 27.3, 25.9,
7.32 (bs, 1H), 7.23–7.20 (m, 1H), 5.64 (s, 1H), 4.17–4.14 (m, 2H), 25.8, 25.6, 25.1, 25.0, 24.9, 24.6, 24.5, 24.0, 21.6, 21.5; LC-MS:
3.70 (s, 3H), 3.67 (bs, 1H), 3.26–3.15 (m, 2H), 2.08–2.06 (m, 1H), 771.41 (M + Na)+, 783.38 (M + K)+; anal. calcd. for C36H56N6O9S:
1.90 (bs, 1H), 1.84–1.82 (m, 2H), 1.75–1.72 (m, 1H), 1.64 (s, 3H), C, 57.73; H, 7.54; N, 11.22; found: C, 57.51; H, 7.38; N, 11.60.
1.61 (bs, 1H), 1.59–1.57 (m, 2H), 1.53 (s, 3H), 1.50 (s, 3H), 1.47 (s,
3H), 1.43 (s, 9H), 0.96–0.95 (d, 3H, J ¼ 6.71 Hz), 0.91–0.89 (m,
tert-Butyl-(4-methyl-1-((2-methyl-1-((2-(((2S)-2-((4-methyl-1-
9H); 13C NMR (100 MHz, CDCl3) d: 174.7, 173.0, 171.5, 156.3,
((2-methyl-1-(methylamino)-1-oxopropan-2-yl)amino)-1-
137.3, 134.9, 129.8, 123.9, 123.2, 123.0, 79.9, 61.9, 57.5, 56.2,
53.4, 52.4, 49.8, 40.0, 31.0, 28.2, 26.1, 25.1, 24.9, 24.8, 24.7, 24.4,
24.1, 23.0, 22.8, 22.1; LC-MS: 803.40 (M + Na)+; anal. calcd. for
oxopentan-2-yl)carbamoyl)pyrrolidin-1-yl)sulfonyl)phenyl)-
amino)-1-oxopropan-2-yl)amino)-1-oxopentan-2-yl)
carbamate 9
C
37H60N6O10S: C, 56.90; H, 7.74; N, 10.76; found: C, 56.63.; H,
Representative procedure. To the ester 9 (0.15 g, 0.02 mmol),
a saturated solution of methylamine in methanol was added at
ꢀC and stirred for 12 h. The solvent was then removed to
obtain the methyl amide 14 as a white solid. Yield: 0.13 g (90%);
m.p.: 203–205 ꢀC; [a]D25: ꢁ55.55ꢀ (c 1.56, CHCl3); IR (CHCl3) n
(cmꢁ1) 3615, 3393, 3019, 2400, 1674, 1523, 1421, 1338, 1215,
1046, 759, 669; 1H NMR (400 MHz, CDCl3) d: 10.15 (s, 1H), 8.63–
7.92.; N, 10.68.
0
Methyl-2-(2-((S)-1-((2-(2-(2-((tert-butoxycarbonyl)amino)-2-
methylpropanamido)-2-methylpropanamido)phenyl)sulfonyl)-
pyrrolidine-2-carboxamido)-4-methylpentanamido)-2-
methylpropanoate 11
Hexapeptide 11 was obtained as a white uffy solid. Yield: 82ꢁ%1; 8.62 (d, 1H, J ¼ 7.93 Hz), 7.79–7.77 (d, 1H, J ¼ 7.93 Hz), 7.72 (s,
m.p.: 93–95 ꢀC; [a]D25: ꢁ101ꢀ (c ¼ 1, CHCl3); IR (CHCl3) n (cm
)
1H), 7.61–7.58 (m, 1H), 7.32 (bs, 1H), 7.23–7.20 (m, 1H), 7.04 (s,
3393, 3019, 2981, 2401, 1725, 1675, 1523, 1294, 1216, 1155, 1H), 6.77 (s, 1H), 5.24 (bs, 1H), 4.25 (bs, 1H), 4.11 (bs, 1H), 4.05–
1
1073, 926, 759, 668; H NMR (400 MHz, CDCl3) d: 9.97 (s, 1H), 4.04 (m, 1H), 3.64 (bs, 1H), 3.22–3.20 (m, 1H), 2.72 (s, 3H), 2.09
8.51–8.49 (d, 1H, J ¼ 8.24 Hz), 7.81–7.79 (d, 1H, J ¼ 7.33 Hz), (bs, 2H), 1.78–1.72 (m, 3H), 1.71–1.64 (m, 3H), 1.61 (s, 3H), 1.57
7.61–7.58 (m, 1H), 7.36 (bs, 1H), 7.24–7.21 (m, 1H), 7.08–7.06 (d, (s, 3H), 1.51 (s, 3H), 1.46 (bs, 2H), 1.41 (s, 3H), 1.39 (s, 9H), 1.02–
1H, J ¼ 8.55 Hz), 5.20 (s, 1H), 4.46–4.41 (m, 1H), 4.19–4.17 (m, 1.01 (m, 3H), 0.94–0.90 (m, 9H); 13C NMR (100 MHz, CDCl3) d:
1H), 3.69 (s, 3H), 3.60 (bs, 1H), 3.18–3.13 (m, 1H), 2.22 (bs, 1H), 174.6, 173.4, 173.1, 172.1, 171.9, 155.7, 137.6, 135.0, 129.6,
2.07–2.04 (m, 1H), 1.91–1.86 (m, 1H), 1.82–1.79 (m, 2H), 1.72– 123.7, 122.9, 122.5, 79.5, 61.4, 57.6, 54.0, 52.6, 50.2, 41.5, 39.5,
1.71 (m, 1H), 1.60 (s, 3H), 1.58 (s, 3H), 1.54–1.52 (m, 1H), 1.50– 30.6, 28.2, 26.5, 25.2, 24.9, 24.5, 24.3, 23.6, 23.0, 22.7, 22.0, 21.7;
1.49 (m, 6H), 1.47 (bs, 6H), 1.42 (s, 9H), 0.94–0.93 (d, 3H, J ¼ LC-MS: 802.41 (M + Na)+, 818.37 (M + K)+; anal. calcd. for
6.41 Hz), 0.91–0.89 (d, 3H, J ¼ 6.41 Hz); 13C NMR (100 MHz,
C37H61N7O9S: C, 56.98; H, 7.88; N, 12.57; found: C, 56.50; H,
CDCl3) d: 174.8, 174.6, 173.1, 171.1, 170.9, 155.1, 137.3, 134.9, 8.02; N, 12.89.
129.7, 123.9, 123.5, 123.0, 80.2, 77.2, 62.1, 57.3, 56.7, 56.1, 52.3,
51.8, 49.6, 40.3, 30.7, 28.2, 25.2, 25.1, 25.0, 24.6, 24.5, 22.9, 21.6;
LC-MS: 775.39 (M + Na)+, 791.30 (M + K)+; anal. calcd. for
tert-Butyl(2-methyl-1-((2-methyl-1-((2-(((2S)-2-((4-methyl-1-((2-
methyl-1-(methylamino)-1-oxopropan-2-yl)amino)-1-
oxopentan-2-yl)carbamoyl)pyrrolidin-1-yl)sulfonyl)phenyl)-
amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)
carbamate 12
C
35H56N6O10S: C, 55.83; H, 7.50; N, 11.16; found: C, 56.03; H,
7.29; N, 11.30.
Methyl-2-methyl-2-(4-methyl-2-(1-((2-(2-methyl-2-((S)-1-
pivaloylpyrrolidine-2-carboxamido)propanamido)phenyl)-
sulfonyl)pyrrolidine-2-carboxamido)pentanamido)
propanoate 14
Compound 12 was obtained as a white solid. Yield: 82%, m.ꢁp1.:
136–138 ꢀC; [a]D25: ꢁ63.52ꢀ (c ¼ 0.85, CHCl3); IR (CHCl3) n (cm
)
3627, 3393, 3019, 2400, 1674, 1522, 1422, 1338, 1215, 1046, 928,
757, 669; 1H NMR (400 MHz, CDCl3) d: 9.93 (s, 1H), 8.50–8.49 (d,
Hexapeptide 14 was obtained as a white uffy solid. Yield: 73%; 1H, J ¼ 8.24 Hz), 7.78–7.77 (m, 1H), 7.63–7.60 (m, 1H), 7.35 (bs,
m.p.: 98–100 ꢀC; [a]D26: ꢁ129.52ꢀ (c ¼ 1.05, CHCl3); IR (CHCl3) n 1H), 7.25–7.20 (m, 2H), 6.82–6.81 (m, 1H), 5.17 (s, 1H), 4.28–4.26
(cmꢁ1) 3333, 3019, 2973, 2400, 1738, 1681, 1584, 1524, 1337, (m, 1H), 4.15–4.13 (m, 1H), 3.66–3.62 (m, 1H), 3.18–3.13 (m,
1215, 1153, 926, 758, 668; 1H NMR (400 MHz, CDCl3) d: 1H), 2.79–2.76 (m, 3H), 2.06–2.02 (m, 2H), 1.94–1.88 (m, 1H),
10.03rotamer (0.8H), 9.96rotamer (0.2H), 8.52–8.50rotamer (0.8H), 1.82–1.79 (m, 2H), 1.75–1.69 (m, 1H), 1.63–1.62 (m, 1H), 1.62 (s,
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3H), 1.60 (s, 3H), 1.51 (s, 6H), 1.50 (s, 3H), 1.49 (s, 3H), 0.98–0.97
(d, 3H, J ¼ 6.41 Hz), 0.93–0.92 (d, 3H, J ¼ 6.41 Hz); 13C NMR (100
MHz, CDCl3) d: 174.9, 174.7, 173.2, 172.1, 171.3, 155.0, 137.3,
134.9, 129.6, 124.0, 123.7, 122.9, 80.2, 77.2, 62.1, 57.5, 57.4, 56.7,
53.1, 49.6, 39.3, 30.7, 28.2, 26.5, 25.7, 25.5, 25.4, 25.3, 25.1, 24.6,
24.5, 22.9, 21.6; LC-MS: 774.45 (M + Na)+, 790.43 (M + K)+; anal.
calcd. for C35H57N7O9S: C, 55.91; H, 7.64; N, 13.04; found: C,
56.13; H, 7.45; N, 13.28.
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(2S)-N-(2-Methyl-1-((2-(((2S)-2-((4-methyl-1-((2-methyl-1-
(methylamino)-1-oxopropan-2-yl)amino)-1-oxopentan-2-yl)
carbamoyl)pyrrolidin-1-yl)sulfonyl)phenyl)amino)-1-
oxopropan-2-yl)-1-pivaloylpyrrolidine-2-carboxamide 15
Compound 15 was obtained as a white solid. Yield: 86%; m.p.:
162–164 ꢀC; [a]D25: ꢁ104ꢀ (c ¼ 1, CHCl3); IR (CHCl3) n (cmꢁ1
)
3615, 3393, 3019, 2400, 1674, 1523, 1421, 1338, 1215, 1046, 928,
767, 669; 1H NMR (400 MHz, CDCl3) d: 9.94rotamer (0.2H),
9.91rotamer (0.8H), 8.43–8.42 (d, 1H, J ¼ 8.31 Hz), 7.79–7.77rotamer
(0.9H), 7.76rotamer (0.7H), 7.67rotamer (0.7H), 7.64rotamer (0.3H),
7.61–7.58 (m, 1H), 7.25–7.22 (m, 1H), 7.07–7.04 (m, 2H), 6.92
(bs, 1H), 4.65–4.62 (m, 1H), 4.35–4.30rotamer (0.8H), 4.28–
4.26rotamer (0.2H), 4.14–4.13rotamer (0.1H), 4.12–4.10rotamer
(0.9H), 3.72–3.59 (m, 3H), 3.16–3.11 (m, 1H), 2.78–2.77rotamer
(2.5H), 2.76rotamer (0.5H), 2.19–2.14 (m, 1H), 2.12–2.08 (m, 1H),
2.04–1.96 (m, 3H), 1.93–1.87 (m, 2H), 1.82–1.77 (m, 2H), 1.73–
1.69 (m, 1H), 1.63–1.58 (m, 1H), 1.55 (s, 3H), 1.54 (s, 3H), 1.53 (s,
3H), 1.50 (s, 3H), 1.20rotamer (2H), 1.16rotamer (7H), 0.99–0.97 (m,
3H), 0.93–0.91 (m, 3H); 13C NMR (100 MHz, CDCl3) d: 178.1,
177.8, 174.9, 174.7, 172.3, 172.2, 172.2, 172.0, 171.7, 137.7,
137.6, 134.9, 129.7, 129.5, 124.3, 124.2, 124.1, 123.4, 61.9, 61.7,
57.6, 57.5, 57.3, 53.1, 52.9, 49.6, 48.3, 40.0, 39.1, 39.0, 30.9, 27.3,
26.5, 25.8, 25.7, 25.6, 25.5, 24.3, 25.1, 25.0, 24.6, 24.5, 23.0, 22.9,
21.6, 21.5; LC-MS: 770.47 (M + Na)+, 786.41 (M + K)+; anal. calcd.
for C36H57N7O8S: C, 57.81; H, 7.68; N, 13.11; found: C, 57.55; H,
7.89; N, 13.35.
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Acknowledgements
AR, ASK and RLG are thankful to CSIR, New Delhi, for a research
fellowship. GJS thanks NCL-IGIB (New Delhi) for nancial
support.
Notes and references
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