Characterization of (Boc-Cys/Sec-NHMe)2 and (Boc-Cys/Sec-OMe)2: Evidence of local conformational difference between disulfide and diselenide

Article abstract of DOI:10.1002/psc.3245

Conformations of disulfide and diselenide were compared in (Boc-Cys/Sec-NHMe)₂ and (Boc-Cys/Sec-OMe)₂ using X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, density functional theory (DFT), and circular dichroism (CD) spectroscopy. Conformations of disulfide/diselenide in polypeptides are defined based on the sign of side chain torsion angle χ₃ (–CH₂–S/Se–S/Se–CH₂–); negative indicates left-handed and positive indicates right-handed orientation. In the crystals of (Boc-Cys-OMe)₂ and (Boc-Sec-OMe)₂, the disulfide exhibits a left-handed and the diselenide a right-handed orientation. Characterization of cystine and selenocystine derivatives in solution using ¹H-NMR, natural abundant ⁷⁷Se NMR, 2D-ROESY, and chemical shift analysis coupled to DMSO titration has indicated the symmetrical nature and antiparallel orientation of Cys/Sec residues about the disulfide/diselenide bridges. Structural calculations of cystine and selenocystine derivatives using DFT further support the antiparallel orientation of Cys/Sec residues about disulfide/diselenide. The far-ultraviolet (UV) region CD spectra of cystine and selenocystine derivatives have exhibited the negative Cotton effect (CE) for disulfide and positive for diselenide confirming the difference in the conformational preference of disulfide and diselenide. In the previously reported polymorphic structure of (Boc-Sec-OMe)₂, the diselenide has right-handed orientation. In the X-ray structures of disulfide and diselenide analogues of Escherichia coli protein encoded by curli specific gene C (CgsC) retrieved from Protein Databank (PDB), disulfide has left-handed and the diselenide right-handed orientation. The current report provides the evidence for the local conformational difference between a disulfide and a diselenide group under unconstrained conditions, which may be useful for the rational replacement of disulfide by diselenide in polypeptide chains.

Full text of DOI:10.1002/psc.3245

Received: 14 September 2019
DOI: 10.1002/psc.3245
Revised: 11 February 2020
Accepted: 11 February 2020
R E S E A R C H A R T I C L E
Characterization of (Boc-Cys/Sec-NHMe) and (Boc-Cys/Sec-
2
OMe) : Evidence of local conformational difference between
2
disulfide and diselenide
1
1
2
Ashwini Dolle | K. Kasi Amarnath Reddy | Shubha Shridhar Gunaga
|
3
3
4
Kiran Krishnamurthy | Dillip Kumar Senapati | Abhijit Rana
|
2
4
3
Kishorkumar Sindogi | Himansu S. Biswal | Srinivasarao Raghothama
|
1
Konkallu Hanumae Gowd
1
Department of Chemistry, School of Chemical
Sciences, Central University of Karnataka,
Kalaburagi, Karnataka, India
Conformations of disulfide and diselenide were compared in (Boc-Cys/Sec-NHMe)
2
and (Boc-Cys/Sec-OMe) using X-ray crystallography, nuclear magnetic resonance
2
2
Solid State and Structural Chemistry Unit
(
(
NMR) spectroscopy, density functional theory (DFT), and circular dichroism
CD) spectroscopy. Conformations of disulfide/diselenide in polypeptides are defined
(
SSCU), Indian Institute of Science, Bangalore,
Karnataka, India
3
NMR Research Centre, Indian Institute of
based on the sign of side chain torsion angle χ (–CH –S/Se–S/Se–CH –); negative
3
2
2
Science, Bangalore, Karnataka, India
4
indicates left-handed and positive indicates right-handed orientation. In the crystals
of (Boc-Cys-OMe) and (Boc-Sec-OMe) , the disulfide exhibits a left-handed and the
School of Chemical Sciences, NISER,
Bhubaneswar, Odisha, India
2
2
diselenide a right-handed orientation. Characterization of cystine and selenocystine
Correspondence
1
77
Dr Konkallu Hanumae Gowd, Department of
Chemistry, School of Chemical Sciences,
Central University of Karnataka, Kalaburagi
derivatives in solution using H-NMR, natural abundant Se NMR, 2D-ROESY, and
chemical shift analysis coupled to DMSO titration has indicated the symmetrical
nature and antiparallel orientation of Cys/Sec residues about the disulfide/diselenide
bridges. Structural calculations of cystine and selenocystine derivatives using
DFT further support the antiparallel orientation of Cys/Sec residues about
disulfide/diselenide. The far-ultraviolet (UV) region CD spectra of cystine and
selenocystine derivatives have exhibited the negative Cotton effect (CE) for disulfide
and positive for diselenide confirming the difference in the conformational
preference of disulfide and diselenide. In the previously reported polymorphic
5
85367, Karnataka, India.
Email: khgowd@cuk.ac.in
Funding information
DST-SERB-ECR, Grant/Award Number:
SERB/ECR/000028
2
structure of (Boc-Sec-OMe) , the diselenide has right-handed orientation. In the
X-ray structures of disulfide and diselenide analogues of Escherichia coli protein
encoded by curli specific gene C (CgsC) retrieved from Protein Databank (PDB), disul-
fide has left-handed and the diselenide right-handed orientation. The current report
provides the evidence for the local conformational difference between a disulfide
and a diselenide group under unconstrained conditions, which may be useful for the
rational replacement of disulfide by diselenide in polypeptide chains.
0
0
0
0
77
ABBREVIATION: (Boc-Cys/Sec-NHMe)
2
, S,S /Se,Se -bis (Boc-Cys/Sec-NHMe); (Boc-Cys/Sec-OMe)
2
, S,S /Se,Se -bis (Boc-Cys/Sec-OMe); Se NMR, selenium-77 nuclear magnetic resonance;
CD Spectroscopy, circular dichroism spectroscopy; CSD, Cambridge Structural Database.
J Pep Sci. 2020;e3245.
wileyonlinelibrary.com/journal/psc
© 2020 European Peptide Society and John Wiley & Sons, Ltd.
1 of 11
https://doi.org/10.1002/psc.3245

2
of 11
DOLLE ET AL.
1
|
INTRODUCTION
the selenocystine derivatives positive bands confirming the difference
in conformational preference of disulfides and diselenides in solution.
The current report has provided the evidence for the local conforma-
tional difference between cysteine disulfides and selenocysteine dis-
elenides under unconstrained structural condition with the disulfides
preferring a left-handed and diselenides a right-handed orientation.
The replacement of disulfide with diselenide bridges is becoming
increasingly common owing to the advantages of selenium as a heavy
1-4
atom in X-ray diffraction, in directing oxidative folding in peptides
4-13
and proteins containing multiple disulfides
and in permitting direct
77
visualization by selenium-77 nuclear magnetic resonance
(
Se
14
NMR) (notwithstanding the fact that such NMR studies of Se-
containing peptides are challenging because of low sensitivity). The
deviations in internal coordinates resulting from substitution of disul-
fide with diselenide are minimal due to similar atomic size (S: 1.02 Å;
Se: 1.17 Å), bond length (S–S: 2.03 Å; Se–Se: 2.33 Å), and electroneg-
2
|
MATERIALS AND METHODS
| Materials and methods
2.1
4,6
ativity (S: 2.58; Se: 2.33). The replacement of disulfide by diselenide
is considered as isosteric in nature due to retention of bioactivities
and overall 3D structures in series of conotoxins and hor-
Cystine and HBTU were purchased from Himedia, India.
Selenocystine, Boc-anhydride, DIPEA, and methylamine hydrochloride
2
(NH Me.HCl) were obtained from Sigma-Aldrich Co, India. Solvents
8
,9,12,13,15-20
mones.
However, local differences in structural features
like 1,4-dioxane, dry tetrahydrofuran (THF), and ethyl acetate were
purchased from Sigma-Aldrich Co. Common chemicals like sodium
were observed due to replacement of Cys1-Cys11 with Sec1-Sec11
1
3
10
in apamin,
Cys2-Cys19 with Sec2-Sec19 in EETI-II,
and
chloride (NaCl), anhydrous sodium sulfate (Na
(NaOH), sodium carbonate (Na CO ), and hydrogen chloride (HCl)
were purchased from SD Fine chemicals. CD OH and D O were
2 4
SO ), sodium hydroxide
19
CysA7-CysB7 with SecA7-SecB7 in bovine insulin. Interestingly, the
2
3
crystal structures of human insulin and Escherichia coli CgsC protein
3
2
0
have revealed the deviation in side chain torsion angle χ
3
and χ
1
by
obtained from Cambridge Isotope Laboratories, Inc, UK, and high-
performance liquid chromatography (HPLC) graded solvents obtained
from Merck, India. Specific rotations of the synthetic products were
recorded with a EQ-801 Equiptronics digital polarimeter equipped
with 2-dm path length, at 589-nm wavelength and at room tempera-
ture in methanol. Mass spectra were recorded by HCT ultra PTM Dis-
covery System ETDII ion trap mass spectrometer (Bruker Daltonics,
Germany).
ꢀ
ꢀ
20-22
2
0
to 140 due to the replacement
of disulfide with diselenide.
Restrictions may have imposed in highly cross-linked polypeptides to
8
,19,23
accommodate a similar conformation for disulfide and diselenide
even though a difference exists in the rotational energy barrier
19,24
between disulfide and diselenide.
Unlike the peptides containing
multiple intramolecular disulfide crosslinks, simple cystine and
selenocystine derivatives may impose less restriction on free rotation
of disulfide/diselenide and serve as good models to access the confor-
mational preference of disulfide and diselenide bridges.
The conformation of cystine in polypeptides is defined by the sign
2.2
|
Synthesis of (Boc-Cys/Sec-NHMe) and (Boc-
2
0
0
of the five side chain torsion angles χ
, χ
1 2
, χ
3
, χ
2
, and χ
1
with sign of
Cys/Sec-OMe)2
χ
3
torsion angle deciding the orientation of disulfide as left handed
23,25,26
(
χ
3
: −ve) or right handed (χ
mational features of disulfide/diselenide bridges were compared in
Boc-Cys/Sec-NHMe) and (Boc-Cys/Sec-OMe) using X-ray crystal-
3
: +ve).
In the current report, confor-
2.2.1
|
Synthesis of (Boc-Cys/Sec-NHMe)2
(
2
2
2
(Boc-Cys/Sec-OH) has been synthesized using the standard proce-
lography, NMR spectroscopy, density functional theory (DFT), and cir-
cular dichroism (CD) spectroscopy. The conformation of diselenide
was also analyzed in the polymorphic structure reported by Singh
dures of synthesis of Boc-protected amino acids. (Boc-Cys/Sec-OH)
2
was dissolved in dry THF, 2 equiv. of HBTU were added, and the solu-
tion was stirred for 5 minutes; then 2.5 equiv. of DIPEA and methyl-
2
7
et al for (Boc-Sec-OMe)
2
. Moreover, conformations of disulfide and
amine hydrochloride (NH
was stirred overnight, extracted with ethyl acetate; the organic layer
was washed with 0.5-N HCl and 1-N Na CO , brined, and dried over
Na SO . Solution was evaporated to dry white powder (80%-90%) of
(Boc-Cys/Sec-NHMe) . The crude product was purified by column
2
Me.HCl) were added. The reaction mixture
diselenide groups were also compared in the X-ray structures of E coli
CgsC protein retrieved from PDB containing unconstrained dis-
ulfide/diselenide bridges where the disulfide exhibits a left-handed
2
3
2
4
21,22
and the diselenide the right-handed orientation.
Conformational
2
analysis of cystine and selenocystine derivatives using NMR spectros-
copy and DFT has revealed similar structural features for the
corresponding derivatives with antiparallel orientation of Cys/Sec res-
idues about the disulfide/diselenide bridges. The absence of aromatic
residues in designed compounds have permitted unambiguous assign-
ment of the handedness of disulfide/diselenide based on the sign of
Cotton effect (CE) in the far-ultraviolet (UV) region of the CD spec-
chromatography over 60 to 120 mesh silica gel with 30% to 40% ethyl
acetate in hexane and further characterized by thin-layer chromatog-
raphy using 40% ethyl acetate-hexane solvent system. The yield and
ꢀ
melting point of (Boc-Cys-NHMe)
2
were 71% and 166 C and of
ꢀ
(Boc-Sec-NHMe)
2
44% and 140 C, respectively. Calculated
+
and observed mass of (Boc-Cys-NHMe)
2
: [M + H] cal. = 467.0 Da,
+
and [M
+
H] obs.
=
2
467.2 Da; and of (Boc-Sec-NHMe) :
2
8-32
+
+
tra.
Similarly, the far-UV CD spectra of the cystine derivatives of
[M + H] cal. = 563.0 Da, and [M + H] obs. = 563.6 Da (for mass spectra,
see Figures S1 and S2 and for NMR data, Table S3). The specific
the present study were found to exhibit negative bands and those of

DOLLE ET AL.
3 of 11
ꢀ
rotation value in methanol for (Boc-Cys-NHMe)
2
is +75 and for (Boc-
spectra were recorded for cystine and selenocystine derivatives at a
ꢀ
Sec-NHMe)
2
−100 .
3
concentration of approximately 3mM in CD OH solution. Spectra
were referenced with solvent peak resonance. Methyl protons of
methanol were set at 3.3100 ppm. Two dimensional TOCSY and
ROESY spectra were recorded in phase-sensitive mode using
STATES-TPPI method. The spectral width of 12 ppm in both dimen-
sions was used with 2048 and 400 time domain points in t2 and t1
domain, respectively. Mixing time of 100 and 250 milliseconds was
used for TOCSY and ROESY spectra, respectively. A relaxation delay
of 1.5 seconds was used throughout 1D and 2D experiments. Signal
averaging was done over 64 scans. During data processing, time
domain points in t1 were zero filled by another 600 points to yield a
final data set of 2K X 1K points before Fourier transformation. Linear
prediction up to 512 points was also used in the t1 domain.
Apodization by shifted sine bell (π/2) was also carried out on both
dimensions. Hydrogen bonding tendency of amide protons of cystine
and selenocystine derivatives was determined by carrying out
2
.2.2
|
Synthesis of (Boc-Cys/Sec-OMe)2
2
To a solution of (Boc-Cys/Sec-OH) in dry THF, 2 equiv. of HBTU
were added, and after 5-min stirring, 2.5 equiv. of DIPEA and HPLC
grade methanol were added. The reaction mixture was stirred over-
night and extracted with ethyl acetate; the organic layer was washed
with 0.5-N HCl and 1-N Na
solution was evaporated to dry white powder (80%-90%) of (Boc-
Cys/Sec-OMe) . The crude product was purified by column chroma-
2 3 2 4
CO , brined, and dried over Na SO . The
2
tography over 60 to 120 mesh silica gel with 30% to 40% ethyl ace-
tate in hexane and further characterized by thin-layer
chromatography using 40% ethyl acetate-hexane solvent system. The
ꢀ
yield and melting point of (Boc-Cys-OMe)
2
were 75% and 92 C and
ꢀ
of (Boc-Sec-OMe)
2
46% and 84 C, respectively. The calculated and
3
DMSO/CDCl solvent titration experiments. The changes in the
+
observed mass of (Boc-Cys-OMe)
2
: [M + H] cal. = 469.0 Da, and
chemical shifts of amide protons were monitored as the function of
+
+
[
M + H] obs. = 469.2 Da; and (Boc-Sec-OMe)
2
: [M + H] cal. = 565.0 Da,
volume of DMSO-d
6
solvent.
77
+
and [M + H] obs. = 565.6 Da (for mass spectra, see Figures S1 and S2
Natural abundant Se-NMR spectra were recorded at 298 K on
and for NMR spectra, Table S3). The specific rotation value in metha-
Bruker Avance-III 400-MHz spectrometer operating at 76.306 MHz
77
ꢀ
ꢀ
nol for (Boc-Cys-OMe)
2
is +75 and for (Boc-Sec-OMe)
2
–125 .
for Se using a 5-mm broadband BBFO probe equipped with a z gra-
31
dient. The inner coil was optimized for observing nuclei between
P
15
1
and
selenocystine derivatives were dissolved in 500 μL of CD
were referenced with diphenyl diselenide at 461.625 ppm for Se-
N and that of outer was optimized for H; 25 mg of
2
.3
|
X-ray crystallography
3
OH. Spectra
77
1
77
Single crystals of (Boc-Cys-OMe)
2
were grown by slow evaporation in
NMR chemical shift. 2D H– Se HMQC spectrum of selenocystine
7
7
hexane and ethyl acetate. Similarly, single crystals of (Boc-Sec-OMe)
2
derivatives at natural abundant Se atom was acquired using stan-
dard gradient selected pulse program. The spectrum recorded using
32 transients for each of 400 t1 increment points using a spectral
were grown in methanol and water system. Good-quality single crys-
tals were collected under an optical microscope equipped with polar-
izer. A high-resolution data were collected on an Oxford Xcalibur
1
77
width of 4807.692 Hz for H and 61069.520 Hz for Se. The recycle
7
7
1
(
Nova) diffractometer equipped with an Eos CCD detector using
delay was set to 2.0 for Se and H detected HMQC experiments.
The raw data were processed using apodization by shifted sine bell
(π/2) in both the dimensions. Spectra were processed and analyzed
using Bruker Topspin 3.6.
MoKα radiation (λ = 0.71073 Å). The single crystals were affixed to a
Hampton research cryoloop using Paratone-N oil and were cooled
with a liquid nitrogen stream using an Oxford cryosystem nitrogen
gas-stream cooling device. Refinement, integration, and reduction
33
were performed using CrysAlisPro. All nonhydrogen atoms were
refined anisotropically. Hydrogen atoms on heteroatoms were located
by the difference in the electron density maps. All C–H atoms were
fixed geometrically. Structures were solved by direct methods, refined
2.5
peptides
|
Computational analysis of synthetic/database
| Structures calculation by DFT
34
35
using SHELXS97 accessed by the WinGX. The computing molecu-
lar graphics were with Ortep diagram using Mercury-CSD 3.10.3 soft-
ware. The structures were also processed using Maestro, Schrödinger,
2.5.1
Geometry optimizations of cystine and selenocystine derivatives
were carried out using Turbomole quantum chemistry package at
RI-B97-d/aug-cc-pVDZ level. The initial geometries were taken
from the crystal structures and were fully relaxed during the opti-
mization. To confirm the existence of noncovalent interactions, we
performed natural bond orbital (NBO) calculations on the optimized
structures in Gaussian 09. Images of the orbitals were generated
with the program GaussView. The presence of bond critical points
within the molecules were found using atoms in molecules (AIM)
software.
2
019. Cambridge Crystallographic Data Centre (CCDC) deposition
number for (Boc-Cys-OMe) is 1952115 and for (Boc-Sec-OMe)
952116.
2
2
1
2
.4
|
NMR spectroscopy
NMR experiments were performed on Bruker Avance-III 800-MHz
spectrometer equipped with 5-mm triple resonance CryoProbe. NMR

4
of 11
DOLLE ET AL.
2
.5.2
|
Torsion angles of disulfides in
selenocystine derivatives were prepared in MeOH, EtOH, or TFE
(Trifluoroethanol). Similarly, 2mM solutions of cystine and
selenocystine were prepared in 0.1-N HCl. The spectra of the freshly
prepared solutions were recorded between 320 and 200 nm at
0.2-nm intervals with a time constant of 4 seconds at room tempera-
ture. The sample spectra were subtracted from blank solvent and
averaged from three independent scans. The spectra were further
cystine/selenocystine derivatives retrieved from
database
Crystal structures of cystine and cystine derivatives were retrieved
from Cambridge Structural Database (https://www.ccdc.cam.ac.uk/
structures). A total of 27 crystal structures were retrieved from the
0
0
database. The torsion angles of disulfides (χ
1
, χ
2
, χ
3
, χ
2
, and χ
1
) were
baseline corrected and smoothed. The molar ellipticity [θ]
M
was
3
6
expressed as unit deg cm dmol−1.
2
measured using Mercury (3.7) software. The nomenclature pro-
posed by Schmidt et al was adopted for assigning the conformation of
26
a disulfide based on the torsion angles.
The nomenclature of the disulfide conformation was extended to
diselenide due to their isosteric nature.
3
3
|
RESULTS
| Characterization of the (Boc-Cys-OMe) and
.1
2
(
Boc-Sec-OMe) conformations in crystals by X-ray
2
2
.5.3
|
Structural analysis of proteins retrieved
crystallography
from PDB
2
Figure 1 shows the crystal structures of (A) (Boc-Cys-OMe) and
The disulfide and diselenide analogues of the E coli CsgC protein were
retrieved from PDB and processed using Maestro, Schrödinger, 2019.
Superposition of two proteins was achieved independently using
SuperPose (http://superpose.wishartlab.com), PyMOL software, and
Maestro, Schrödinger, 2019. The nomenclature proposed by Govindu
et al was adopted for assigning the conformation of dis-
(B) (Boc-Sec-OMe)
ulfide/diselenide in (Boc-Cys-OMe)
ulfide/diselenide were compared in the crystals of (Boc-Cys-OMe)
and (Boc-Sec-OMe) with respect to bond lengths, bond angles,
and torsion angles (Figure 1 and Table 1). The deviation in bond
length of S–S/Se–Se is ±0.26 and that of –S/C –Se is
–S–S
2
. Table 1 shows the internal coordinates of dis-
2
and (Boc-Sec-OMe) Dis-
2
.
2
2
Å
C
β
β
23
ulfide/diselenide bridges based on the torsion angles in proteins.
±0.15 Å. Similarly, the deviation in bond angles between –C
β
ꢀ
−/–S–S–C
β
– and –C
β
–Se–Se−/–Se–Se–C
β
– is ±3.09 . These devia-
tions are consistent with previous reports on replacement of cys-
6
,8,19,20
2
.6
|
Circular dichroism
tine with selenocystine in polypeptides.
of disulfide/diselenide were analyzed in the crystals based on the
symmetrical criterion of (Boc-Cys-OMe) and (Boc-Sec-OMe) about
disulfide/diselenide. The signs of χ , χ torsion
The torsion angles
The CD spectra were recorded on a JASCO spectropolarimeter using
cell of 1-mm path length; 2mM solutions of cystine and
2
2
0
0
1
a
1
2
, χ
3
, χ
2
, and χ
FIGURE 1 Crystal structures
of (A) (Boc-Cys-OMe) and
B) (Boc-Sec-OMe)
2
(
2

DOLLE ET AL.
5 of 11
TABLE 1 Internal coordinates of disulfide and diselenide derived
orientation of diselenide is retained in both the crystal forms of (Boc-
from the X-ray structure of (Boc-Cys-OMe)
2
and (Boc-Sec-OMe)
2
2
Sec-OMe) .
Properties
(Boc-Cys-OMe) (Boc-Sec-OMe)
2
2
3
.2
|
Characterization of conformation of (Boc-
Bond length (Se–Se) ( Å)
Cα–Cβ–S/Cα–Cβ–Se
Cβ–S–S/Cβ–Se–Se
2.0
112.2
101.7
−79.9
−76.4
−70.7
−60.0
−70.5
2.3
112.3
101.01
−62.7
−38.3
99.0
Cys/Sec-NHMe) and (Boc-Cys/Sec-OMe) in solution
by NMR spectroscopy
2
2
χ
χ
χ
χ
χ
1
2
3
2
1
The conformations of (Boc-Cys/Sec-NHMe)
2
and (Boc-Cys/Sec-
1
77
2 3
OMe) in CD OH were characterized using 1D- H and Se NMR,
1
1
77
H-homonuclear 2D-TOCSY, 2D-ROESY, and H– Se heteronuclear-
HMQC experiments. Hydrogen bonding of amide protons in cystine
and selenocystine derivatives was determined by measuring the
dependence of amide proton chemical shifts on concentration of
0
0
102.8
−176.4
DMSO using DMSO-CDCl
3
solvent titration experiments. Figure S5
, (b) (Boc-
. Table S3
provides the chemical shift values for protons of cystine and
1
angles of disulfide in (Boc-Cys-OMe)
2
are −, −, −, −, and −, which
shows 800-MHz H-NMR spectra of (a) (Boc-Cys-NHMe)
2
correspond to −LHSpiral conformation. Similarly, the signs of χ
1
, χ
2
,
2 2 2
Sec-NHMe) , (c) (Boc-Cys-OMe) , and (d) (Boc-Sec-OMe)
0
0
χ
3
, χ
2
, and χ
1
torsion angles of diselenide in (Boc-Sec-OMe)
2
are
7
7
−
, −, +, +, and − corresponding to −RHHook conformation. The
is left handed and that
is right handed.
The Cys residues in (Boc-Cys-OMe) were found to exhibit the
following ϕ and ψ values: −94.92 (ϕ) and −174.39 (ψ) in chain A
Figure 1A) and −95.23 (ϕ) and 166.03 (ψ) in chain B (Figure 1A), while
the Sec residues in (Boc-Sec-OMe) have the following ϕ and ψ
values: −117.26 (ϕ) and 60.29 (ψ) in chain A (Figure 1B) and −76.39
ϕ) and 161.72 (ψ) in chain B (Figure 1B). As shown in Figure 1, in the
structures of (Boc-Cys-OMe) and (Boc-Sec-OMe) , the two Cys/Sec
selenocystine derivatives and Se for the selenocystine derivatives.
The protons and selenium resonance peaks in NMR spectra of cystine
and selenocystine derivatives correspond to only half-cystine/half-
selenocystine derivatives indicating similar chemical environment on
either side of the disulfide/diselenide bridges. Cystine and
selenocystine derivatives were further characterized using 2D-TOCSY
(Figure S6) and 2D-ROESY spectra (Figure 2). The observed Nuclear
Overhauser Effects (NOEs) between Boc protons and –NHMe amide
orientation of disulfide in (Boc-Cys-OMe)
2
of diselenide in (Boc-Sec-OMe)
2
2
(
2
(
proton of (Boc-Cys-NHMe)
2
(Figure 2A) and Boc protons and –NHMe
(Figure 2B)
2
2
amide as well as the methyl protons of (Boc-Sec-NHMe)
2
residues display an equiverse orientation with respect to the dis-
ulfide/diselenide groups. Figure S3 shows the packing of molecules in
together with observed through bond correlation in 2D-TOCSY
(Figures S6a and S6b) confirm the antiparallel orientation of Cys/Sec
residues about the disulfide/diselenide groups. Similarly, the observed
the unit cells of (Boc-Cys-OMe)
vides the crystal parameters of (Boc-Cys-OMe)
In the unit cell of (Boc-Cys-OMe) , the molecules are arranged later-
2
2
and (Boc-Sec-OMe) . Table S1 pro-
2
and (Boc-Sec-OMe)
2
.
NOEs between Boc protons and –OMe protons in (Boc-Cys-OMe)
2
2
(Figure 2C) and (Boc-Sec-OMe) (Figure 2D) together with observed
2
ally and stabilized by the intermolecular hydrogen bonding with 2.2-Å
through bond correlation in 2D-TOCSY (Figure S6c,d) confirm the
antiparallel orientation of Cys/Sec residues about the dis-
ulfide/diselenide. Figure S7 shows chemical shift dependence of
distances between –NH and –CO groups (Figure S3a). Similarly, in the
2
unit cell of (Boc-Sec-OMe) , the molecules are arranged horizontally
and stabilized by the hydrophobic interactions involving Boc and
OMe groups (Figure S3b).
amide protons of (a) (Boc-Cys-NHMe)
2
,
(b) (Boc-Sec-NHMe)
2
,
(c) (Boc-Cys-OMe) , and (d) (Boc-Sec-OMe)
2
2
in the DMSO-CDCl sol-
3
Singh et al have reported a different crystal form of (Boc-Sec-
vent titration experiment. It is evident from Figure S7a,b that the
amide proton (–NH) chemical shift of cystine/selenocystine residue is
significantly downfield shifted and that of corresponding –NH of –
NHMe is nearly constant with addition of DMSO indicating that only
27
OMe)
In the current report, (Boc-Sec-OMe)
rhombic form from MeOH and H O. The crystal structure reported by
Singh et al was designated as Form-I and that of current study as
Form-II. Figure S4 shows structure of (Boc-Sec-OMe) in the two dif-
2
, which is a monoclinic form crystalized from petroleum ether.
2
was crystalized in the ortho-
2
27
the –NH of –NHMe is hydrogen bonded in (Boc-Cys-NHMe)
2
/(Boc-
2
Sec-NHMe)
2
.
ferent crystal forms along with superposed structures. The orientation
of Sec residues differs in Form-I and Form-II about the diselenide.
Table S2 shows the comparison of internal coordinates of diselenide
It is also evident from Figure S7c,d that the –NH of
cystine/selenocystine residues are not hydrogen bonded in (Boc-Cys-
OMe)
features for antiparallel orientation of peptide chains about the disul-
fide in (Boc-Cys-Ala-OMe) using DMSO-CDCl solvent titration
experiments. Interestingly, the DMSO titration profiles are very sim-
ilar between (Boc-Cys-NHMe) and (Boc-Sec-NHMe) as well as
and (Boc-Sec-OMe) . Figure S8 shows
and (Boc-Sec-OMe)
2 2
and (Boc-Sec-OMe) . Raj et al have reported similar structural
in the two different crystal forms of (Boc-Sec-OMe)
ferences between the two forms exist at the torsion angles of dis-
elenide and the Ramachandran angles. In Form-I of (Boc-Sec-OMe)
2
. The striking dif-
2
3
32
2
,
0
0
the signs of χ
1
, χ
2
, χ , χ
3 2
, and χ
1
torsion angles of diselenide are −, −,
2
2
+
, +, and + corresponding to ±RHHook conformation. The sign of
between (Boc-Cys-OMe)
2
2
0
77
peripheral χ
1
torsion angle of the diselenide is reversed between two
However, the right-handed
1D- Se NMR spectrum of (Boc-Sec-NHMe)
2
2
.
crystal forms of (Boc-Sec-OMe)
2
.
The observed single chemical shift for selenium supports the

6
of 11
DOLLE ET AL.
FIGURE 2 2D-ROESY spectrum of (A) (Boc-Cys-NHMe)
2
, (B) (Boc-Sec-NHMe)
2
, (C) (Boc-Cys-OMe)
2
, and (D) (Boc-Sec-OMe)
2
in methanol.
The observed NOEs are encircled. In case of (D), spectra are shown from 0 to 5 ppm to highlight the observed NOE between Boc and OMe
groups
symmetrical nature of diselenide in selenocystine derivatives. Figure 3
calculations by DFT—(a) symmetrical nature of the Cys/Sec residues
about disulfide and diselenide, (b) NOEs between Boc methyl protons
and –NH of –NHMe and methyl protons of –NHMe or Boc methyl
protons and methyl protons of OMe, (c) intramolecular hydrogen
1
77
shows natural abundance H– Se HMQC spectrum of (Boc-Sec-
NHMe)
2
2
and (Boc-Sec-OMe) . The observed cross peaks between Se
β
atom and H protons of the same selenocysteine residue further con-
firms the symmetry of diselenide in (Boc-Sec-NHMe)
OMe) . In the case of unsymmetrical diselenides, a different set of
cross peaks have been detected between Se and C H at different
2
and (Boc-Sec-
bonding of –NH of –NHMe in (Boc-Cys/Sec-NHMe)
structural features between (Boc-Cys-NHMe) and (Boc-Sec-NHMe)
as well as (Boc-Cys-OMe) and (Boc-Sec-OMe)
DFT optimized structures for (A) (Boc-Cys-NHMe)
NHMe) , (C) (Boc-Cys-OMe)
reports the conformations of disulfide/diselenide in the optimized
lowest energy conformers of (Boc-Cys/Sec-NHMe) and (Boc-
Cys/Sec-OMe) . The conformations of disulfide/diselenide in the opti-
mized structures are as follows: (Boc-Cys-NHMe) is −RHStaple;
(Boc-Sec-NHMe) is −RHStaple; (Boc-Cys-OMe) is +RHSpiral; (Boc-
Sec-OMe) is −RHStaple. The energy difference between the lowest
2
, and (d) similar
2
2
2
β
2
2
. Figure 4 shows the
77
Se signals as revealed from studies on [Sec13 and Sec14] analogue
2
, (B) (Boc-Sec-
14
of κ-ACTX-Hv1c. The current NMR studies support similar struc-
tural features for (Boc-Cys-NHMe) and (Boc-Sec-NHMe) as well as
for (Boc-Cys-OMe) and (Boc-Sec-OMe) in the solution.
2
2 2
, and (D) (Boc-Sec-OMe) . Table S4
2
2
2
2
2
2
2
3
.3
|
DFT studies on the solution structures of
2
2
(
Boc-Cys/Sec-NHMe) and (Boc-Cys/Sec-OMe)2
2
2
energy conformers among the given structures varies by 2.6 to
34.1 kJ/mol (Table S4). The disulfide and diselenide prefer the right-
handed orientation in the optimized structure of cystine/selenocystine
The following structural features of cystine/selenocystine derivatives
derived from NMR studies were used as inputs for structure

DOLLE ET AL.
7 of 11
(
Boc-Cys/Sec-NHMe)
2
(Figure 4A,B) include antiparallel orientation of
Cys/Sec residues about disulfide/diselenide and intramolecular hydro-
gen bonding of 1.9 Å between methylamide –NH and Boc carbonyl
about disulfide/diselenide. Similarly, structural features of (Boc-
2
Cys/Sec-OMe) (Figure 4C,D) include antiparallel orientation of
Cys/Sec residues about disulfide/diselenide. NBO calculations were
performed to confirm the existence of hydrogen bonding and n ! π*
interaction between sulfur/selenium of disulfide/diselenide and neigh-
boring carbonyl group in cystine/selenocystine derivatives. The calcu-
lations have confirmed the presence of hydrogen bonding in (Boc-
Cys/Sec-NHMe)
2
and the existence of n ! π* interaction in (Boc-
Cys/Sec-OMe) . Figure S9 shows a representative example of NBO
2
indicating an overlap of nonbonding orbital of sulfur (n) and π* anti-
bonding orbital of carbonyl and a molecular graph indicating the pres-
ence of BCPs in the path of n ! π* interaction in (Boc-Cys-OMe)
2
. In
case of n ! π* interaction, sulfur/selenium donates its n-orbital elec-
tron density into the π* antibonding orbital of a neighboring carbonyl
37
group. These interactions contribute to the stability of conformation
of (Boc-Cys-OMe) and (Boc-Sec-OMe)
2
2
.
3
.4
|
Characterization of the (Boc-Cys/Sec-
NHMe) and (Boc-Cys/Sec-OMe) conformations in
2
2
solution by CD spectroscopy
Figure 5 shows the CD spectra of (A) (Boc-Cys-NHMe)
2
/(Boc-Sec-
NHMe) and (B) (Boc-Cys-OMe) /(Boc-Sec-OMe) in methanol, etha-
2
2
2
nol, and trifluroethanol. The positive band at approximately 220 nm
and negative band at approximately 200 nm indicate the random coil
nature of cystine and selenocystine derivatives in solution. The region
from 250 to 320 nm has been widely used to investigate the confor-
1
77
FIGURE 3 Natural abundant H– Se HMQC spectrum of
A) (Boc-Sec-NHMe) and (B) (Boc-Sec-OMe)
(
2
2
3
0,31,38
derivatives. However, in the case of (Boc-Cys-NHMe)
ately following left-handed conformer differs by 33.8 kJ/mol and in
that of (Boc-Cys-OMe) by 2.6 kJ/mol. The structural features of
2
, the immedi-
mation of disulfides in polypeptides.
These regions are ampli-
fied in Figure 5A,B for the corresponding cystine and selenocystine
derivatives. As indicated in Figure 5A,B, the cystine derivatives exhibit
2
FIGURE 4 The density
functional theory (DFT) optimized
structures of (A) (Boc-Cys-NHMe)
2
,
(
2
B) (Boc-Sec-NHMe) , (C) (Boc-Cys-
OMe) , and (D) (Boc-Sec-OMe)
2
2

8
of 11
DOLLE ET AL.
2 2
FIGURE 5 The circular dichroism (CD) spectra of (A) (Boc-Cys/Sec-NHMe) and (B) (Boc-Cys/Sec-OMe) . The far-UV region shown in the
rectangular box was amplified in corresponding panels
negative CE at approximately 260 nm and the selenocystine deriva-
tives positive CE at approximately 280 nm. The CE of diselenide is red
shifted by approximately 15 nm compared with that of disulfide.
These observations are consistent with previous CD reports on dis-
negative disulfide CD band and (Boc-Sec-OMe)
elenide CD band. As shown in Figure 5A,B, (Boc-Cys-OMe)
(Boc-Sec-OMe) have indeed yielded negative disulfide and positive
diselenide CD bands, respectively. It has been reported that cystine
2
to a positive dis-
2
and
2
29,39
2
−1
ulfides and diselenides.
Interestingly, the sign of CE is reversed
with [θ]
M
~ ±2000 deg cm dmol indicates the presence of mixtures
between disulfide and diselenide in the cystine and selenocystine
derivatives. On the contrary in the CD spectra of cystine and
selenocystine in 0.1-N HCl (Figure S10), both disulfide and diselenide
have negative CEs. These results are consistent with previous CD
of left-handed (M) and right-handed (P) disulfide conformers in solu-
31
tion. The occurrence of CD signal in such solutions may indicate the
presence of enantiomeric excess of specific disulfide conformation in
the solution. Therefore, the negative CD band of the disulfide of cys-
tine derivatives (Figure 5) may indicate the presence of an excess of
left-handed (M) disulfide conformer in solution. Similarly, positive CD
band of diselenide of selenocystine derivatives (Figure 5) may indicate
the presence of excess of right-handed (P) diselenide conformer in
solution. The following conclusion can be drawn from the change in
the sign of CD signals between cystine and selenocystine derivatives:
(a) Handedness is reversed between disulfide and diselenide, (b) large
change in torsion angle with retention of chirality in diselenide, or
(c) difference in enantiomeric excess of disulfide and diselenide con-
formers. The CD studies confirmed the difference in conformational
preference of disulfide and diselenide of cystine and selenocystine
derivatives in the solution.
38
40
41
reports by Casey and Martin, Ringdahl et al, and Djerassi et al
for cystine and selenocystine. The correlation of the sign of CD bands
of disulfide with the chirality can be made using quadrant rule and its
30,31
modified version.
Particularly, in the absence of aromatic residues,
the sign of CD band can be directly correlated to the torsion angle χ
3
30,42
(
–CH
2
–S–S–CH
2
–) using quadrant rule.
Since the chirality of
disulfide and diselenide arises due to helicity, the quadrant rule of cys-
2
9,39
teine disulfide can be extended to selenocysteine diselenide.
As
has χ
value
determined from crystals, the disulfide in (Boc-Cys-OMe)
2
3
ꢀ
value of −70.6 and that of diselenide in (Boc-Sec-OMe)
2
has χ
3
ꢀ
of +99 . Assuming that major deviations from these values did not
2
occur in solution, it is expected that (Boc-Cys-OMe) gives rise to

DOLLE ET AL.
9 of 11
4
|
DISCUSSION
CE and the diselenide of (Boc-Sec-NHMe/OMe)
2
a positive CE. These
results further support the observed conformational difference
between disulfide and diselenide in crystal structures of (Boc-Cys-
The conformations of disulfide and diselenide were compared in (Boc-
Cys/Sec-NHMe) and (Boc-Cys/Sec-OMe) . In crystals of (Boc-Cys-
OMe) and (Boc-Sec-OMe) , the disulfide has −LHSpiral and dis-
elenide the −RHHook conformation. However, crystals of (Boc-Cys-
OMe) exhibit a C2 space group and those of (Boc-Sec-OMe)
P22 space group. In the independent studies, Singh et al have
reported the polymorphic form of (Boc-Sec-OMe) with a C2 space
2
2
2 2
OMe) and (Boc-Sec-OMe) . In contrast, in disulfide and diselenide
8
19
2
2
analogue crystals of α-conotoxin, bovine insulin,
and human
20
insulin, the handedness is retained between disulfide and diselenide
19
2
2
a
(Table S5). However, in bovine insulin, even though handedness is
retained between disulfide and diselenide, subtle structural differ-
ences are apparent around disulfide/diselenide. The local conforma-
tional differences between disulfide diselenide have been observed in
1 1
2
2
group wherein diselenide exhibits ±RHHook conformation. Interest-
ingly, the crystal structures of the disulfide (PDB ID: 2Y2Y) and dis-
elenide (PDB ID: 2XSK) analogues of E coli CsgC protein have both
the C2 space group (see structures in the PDB). Moreover, the struc-
ture of E coli CsgC protein is same in the free thiol and disulfide form
indicating that the native Cys29-Cys31 disulfide of E coli CsgC is
13
10
solution for apamin and EETI-II. Further, Fiori et al have stated
that the observed local structural difference between apamin with
[Sec1 and Sec11]-apamin is “probably because of the different spatial
requirements of the selenium atoms connecting the two
13
selenocysteines.” The current report provides the evidence for local
conformational difference between disulfide and diselenide under
unconstrained conditions in polypeptide.
21
unconstrained. The disulfide of E coli CsgC has an (+,−)LHStaple
conformation and that of the diselenide analogue the (+,−)
AntiRHHook conformation (Figure 6). These observations suggest
that the disulfide has preference for left-handed orientation and that
of diselenide for right-handed orientation. Similarly, in the far-UV CD
The conformations of disulfide/diselenide were analyzed in the
crystal structures of cystine and cystine derivatives and also of
selenocystine retrieved from Cambridge Structural Database
(Tables S6 and S7). In the 27 crystal forms of cystine and cystine
2
spectra, the disulfide of (Boc-Cys-NHMe/OMe) exhibits a negative
FIGURE 6 Conformational analysis of disulfide and diselenide in the crystal structures of Escherichia coli CsgC retrieved from PDB.
A) Disulfide containing E coli CsgC, (B) diselenide containing E coli CsgC, and (C) superposed structures. Conformation of disulfide and diselenide
are indicated
(

1
0 of 11
DOLLE ET AL.
derivatives, the disulfide is found in six different conformations,
2. Strub MP, Hoh F, Sanchez JF, et al. Selenomethionine and
selenocysteine double labeling strategy for crystallographic phasing.
Structure. 2003;11(11):1359-1367.
namely, +LHSpiral, +RHSpiral, −LHSpiral, −RHSpiral, −LHStaple, and
−RHHook. Interestingly, left-handed and right-handed orientations
3
.
Walter R, Du Vigneaud V. 6-Hemi-L-selenocystine-oxytocin and
-deamino-6-hemi-L-selenocystine-oxytocin, highly potent isologs of
oxytocin and 1-deamino-oxytocin. J am Chem Soc. 1965;87(18):4192-
193.
are equally presented by the disulfide in crystals of cystine and cys-
tine derivatives. In the crystals of cystine and selenocystine
1
4
(
dihydrochloride form), both disulfide and diselenide have the same
4
5
.
.
Moroder L, Musiol H-J. Amino acid chalcogen analogs as tools in pep-
tide and protein chemistry. J Pep Sci. 2019;26(2):1–15.e3232.
Celie PHN, Kasheverov IE, Mordvintsev DY, et al. Crystal structure of
nicotinic acetylcholine receptor homolog AChBP in complex with an
alpha-conotoxin PnIA variant. Nat Struct Mol Biol. 2005;12(7):
left-handed orientation with +LHSpiral conformation. The calculated
energy difference between the left-handed and right-handed dis-
ulfide/diselenide conformers in (Boc-Cys/Sec-NHMe)
2
and (Boc-
Cys/Sec-OMe) using DFT varies by 2.6 to 34.1 kJ/mol. These obser-
2
5
82-588.
Muttenthaler M, Alewood PF. Selenopeptide chemistry. J Pept Sci.
008;14(12):1223-1239.
vations suggest that the conformational change between handedness
of disulfide is subtle in cystine and cystine derivatives. In the case of
selenocystine and selenocystine derivatives, the missing availability
of an adequate number of crystal structures prevents insights on the
propensity for handedness in diselenide. Figure S11 shows mapping
6
.
2
7. Beld J, Woycechowsky KJ, Hilvert D. Chapter 5 Selenocysteine as a
probe of oxidative protein folding. In: Oxidative Folding of Peptides and
Proteins. Cambridge: The Royal Society of Chemistry; 2009:253-273.
8
.
Muttenthaler M, Nevin ST, Grishin AA, et al. Solving the alpha-
conotoxin folding problem: efficient selenium-directed on-resin gen-
eration of more potent and stable nicotinic acetylcholine receptor
antagonists. J am Chem Soc. 2010;132(10):3514-3522.
0
0
of the torsion angles χ
, χ
1 2
, χ
, χ
3 2
, and χ
1
of disulfide/diselenide of
(
Boc-Cys/Sec-OMe) and E coli CsgC on wheel projection. Interest-
2
ingly, the large deviations in the torsion angles between disulfide and
0
9. Walewska A, Zhang M-M, Skalicky JJ, Yoshikami D, Olivera BM,
Bulaj G. Integrated oxidative folding of cysteine/selenocysteine con-
taining peptides: improving chemical synthesis of conotoxins. Angew
Chem Int Ed Engl. 2009;48(12):2221-2224.
diselenide are centered at χ
planes about C
2
, χ
3
, and χ
2
that describes rotations of
bonds encompassing
β
–S/Se, S/Se-S/Se, and S/Se–C
β
sulfur/selenium. In crystals of cystine/selenocystine (dihydrochloride
form), the torsion angles are nearly the same between disulfide and
diselenide, and the only obvious difference is increase in the length
10. Walewska A, Jaskiewicz A, Bulaj G, Rolka K. Selenopeptide analogs of
EETI-II retain potent trypsin inhibitory activities. Chem Biol Drug des.
2
011;77(1):93-97.
43
of the cell edge for selenocystine. However, inspection of struc-
tures in the unit cells have revealed for sulfur a 3.5 Å and for sele-
nium a 3.6-Å distance from chlorine, which corresponds to the sum
of their Van der Waals radius. The minimal deviation in bond lengths
and bond angles due to replacement of disulfide to diselenide in the
currently reported cystine and selenocystine derivatives may contrib-
ute to induce the conformational change between disulfide and
diselenide.
1
1
1
1. Walewska A, Han TS, Zhang M-M, Yoshikami D, Bulaj G, Rolka K.
Expanding chemical diversity of conotoxins: peptoid-peptide chi-
meras of the sodium channel blocker mu-KIIIA and its selenopeptide
analogues. Eur J Med Chem. 2013;65:144-150.
2. Pegoraro S, Fiori S, Cramer J, Rudolph-Bohner S, Moroder L. The
disulfide-coupled folding pathway of apamin as derived from
diselenide-quenched analogs and intermediates. Protein Sci. 1999;
8(8):1605-1613.
3. Fiori S, Pegoraro S, Rudolph-Bohner S, Cramer J, Moroder L. Synthe-
sis and conformational analysis of apamin analogues with natural and
non-natural cystine/selenocystine connectivities. Biopolymers. 2000;
ACKNOWLEDGEMENTS
5
3(7):550-564.
We specially acknowledge Prof T.N. Guru Row, SSCU, IISc, for invalu-
able support with X-ray crystallography of the synthetic peptide. We
acknowledge Dr Govindaraju M., MBU, IISc, for the kind support with
CD spectroscopy. We also thank Yamanappa for insights on NMR
studies. A.D. is supported by the award of Junior Research Fellowship
14. Mobli M, Dantas de Araujo A, Lambert LK, et al. Direct visualization
77
of disulfide bonds through diselenide proxies using Se NMR spec-
troscopy. Angew Chem Int Ed Engl. 2009;48:9312-9314.
1
5. Pegoraro S, Fiori S, Rudolph-Bohner S, Watanabe TX, Moroder L.
Isomorphous replacement of cystine with selenocystine in endothelin:
oxidative refolding, biological and conformational properties of [Sec3,
Sec11,Nle7]-endothelin-1. J Mol Biol. 1998;284(3):779-792.
(
JRF) from the University Grant Commission (UGC), India.
1
6. Gowd KH, Yarotskyy V, Elmslie KS, Skalicky JJ, Olivera BM, Bulaj G.
Site-specific effects of diselenide bridges on the oxidative folding of a
cystine knot peptide, omega-selenoconotoxin GVIA. Biochemistry.
K.K.A.R. acknowledges the JRF fellowship of DST-SERB-ECR grant.
K.H.G. acknowledges the financial support of DST-SERB-ECR
research grant (SERB/ECR/000028). The mass spectrometric data
were acquired from proteomic facility of MBU, IISc, and special thanks
to Mrs Sunita Prakash.
2
010;49(12):2741-2752.
17. Clark RJ, Tan CC, Preza GC, Nemeth E, Ganz T, Craik DJ. Understand-
ing the structure/activity relationships of the iron regulatory peptide
hepcidin. Chem Biol. 2011;18(3):336-343.
1
8. de Araujo AD, Callaghan B, Nevin ST, et al. Total synthesis of the
analgesic conotoxin MrVIB through selenocysteine-assisted folding.
Angew Chem Int Ed Engl. 2011;50(29):6527-6529.
ORCID
Konkallu Hanumae Gowd
https://orcid.org/0000-0002-8991-9368
1
9. Arai K, Takei T, Okumura M, et al. Preparation of selenoinsulin as a
long-lasting insulin analogue. Angew Chem Int Ed Engl. 2017;56(20):
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