The deptq+ experiment: Leveling the dept signal intensities and clean spectral editing for determining chn multiplicities

Article abstract of DOI:10.3390/molecules26123490

We propose a new¹³C DEPTQ⁺ NMR experiment, based on the improved DEPTQ experiment, which is designed to unequivocally identify all carbon multiplicities (Cq, CH, CH2, and CH3) in two experiments. Compared to this improved DEPTQ exper

Full text of DOI:10.3390/molecules26123490

molecules
Article
The DEPTQ+ Experiment: Leveling the DEPT Signal Intensities
and Clean Spectral Editing for Determining CHn Multiplicities
Peter Bigler *, Camilo Melendez and Julien Furrer *
Departement für Chemie, Biochemie und Pharmazie, Universität Bern, Freiestrasse 3, CH-3012 Bern, Switzerland;
camilo.melendez@dcb.unibe.ch
* Correspondence: biglermeier@bluewin.ch (P.B.); julien.furrer@dcb.unibe.ch (J.F.)
Abstract: We propose a new 13C DEPTQ+ NMR experiment, based on the improved DEPTQ experi-
ment, which is designed to unequivocally identify all carbon multiplicities (Cq, CH, CH2, and CH3)
in two experiments. Compared to this improved DEPTQ experiment, the DEPTQ+ is shorter and
1
the different evolution delays are designed as spin echoes, which can be tuned to different JCH
values; this is especially valuable when a large range of 1JCH coupling constants is to be expected.
These modifications allow (i) a mutual leveling of the DEPT signal intensities, (ii) a reduction in J
cross-talk in the Cq/CH spectrum, and (iii) more consistent and cleaner CH2/CH3 edited spectra.
The new DEPTQ+ is expected to be attractive for fast 13C analysis of small-to medium sized molecules,
especially in high-throughput laboratories. With concentrated samples and/or by exploiting the high
sensitivity of cryogenically cooled 13C NMR probeheads, the efficacy of such investigations may be
improved, as it is possible to unequivocally identify all carbon multiplicities, with only one scan, for
each of the two independent DEPTQ+ experiments and without loss of quality.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Keywords: NMR; 13C; DEPTQ; DEPTQ+, spectral editing; J-cross talk; signal intensity leveling
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Bigler, P.; Melendez, C.;
Furrer, J. The DEPTQ+ Experiment:
Leveling the DEPT Signal Intensities
and Clean Spectral Editing for
Determining CHn Multiplicities.
Molecules 2021, 26, 3490. https://
doi.org/10.3390/molecules26123490
1. Introduction
Two-dimensional heteronuclear NMR techniques for 13C assignment have long been
considered extremely powerful since they are much more sensitive than, e.g., one-dimensional
(1D) 13C spectral editing experiments [
], and the corresponding spectra contain a lot of
information. Nevertheless, “old” 1D experiments such as SEFT [ ], APT [ ] and PEN-
1
2
3
Academic Editor: Antony J. Williams
DANT [4,5], refocused variants of INEPT [6], DEPT [7,8], DEPTQ [9–11] and SEMUT [12],
continue to be very useful for routine applications [13]. A likely reason for the popu-
larity of these 1D experiments is the rather low resolution achieved in the indirect di-
Received: 6 May 2021
Accepted: 27 May 2021
Published: 8 June 2021
mension (13C) with 2D heteronuclear experiments, such as HSQC [14], HMBC [15
–17] or
LR-HSQMBC [18], under standard experimental conditions, thus potentially preventing
the unambiguous differentiation between closely spaced 13C resonances. In principle, this
problem can easily be alleviated [19,20] but the required experiments/methods can only be
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iations.
routinely applied to a certain extent, as they require in-depth knowledge of theoretical and
experimental NMR spectroscopy.
One-dimensional 13C experiments such as APT [
2], PENDANT [4,5], and DEPTQ [9–11],
provide information about both quaternary and protonated carbons in the spectra, with
the signals of Cq and CH2 carbons being 180◦ out-of-phase with respect to the signals of
CH and CH3 carbons. Among those experiments, the DEPTQ encompasses all the known
advantages of the basic DEPT experiment but includes the signals of quaternary carbons.
The DEPT pulse sequences, thanks to their marked insensitivity to experimental parameters
compared to other experimental schemes, have proven to be the experiments of choice
Copyright:
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Licensee MDPI, Basel, Switzerland.
This article is an open access article
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Attribution (CC BY) license (https://
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4.0/).
for obtaining the chemical shift and multiplicity information for all types of protonated
1
carbons [
8
]. Additionally, a version of DEPTQ with the length of the initial H pulse
adjusted accordingly allows simplified spectral editing: with this first and the final proton
pulse adjusted to 45◦ and 90◦, respectively, for a DEPTQ90 (DEPTQ9045) and adjusted to
Molecules 2021, 26, 3490. https://doi.org/10.3390/molecules26123490
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3490
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◦
◦
90 and 135 , respectively, for a DEPTQ135 (DEPTQ13590), the intensities of CH signals are
equal in both experiments, since they are reduced to 70% of their maximum value with
the DEPTQ9045 experiment. Thus, subtracting the DEPTQ9045 and the DEPTQ13590 from
each other yields a CH2/CH3 edited spectrum with the signals of the two carbon types in
opposite phase [11]. The DEPTQ90 spectrum shows exclusively, and with opposite phase,
the signals of Cq and CH.
Therefore, although no Cq-. CH-, CH2- and CH3-only spectra result, the pairwise
editing with opposite signs for the signals of different multiplicities allows unambiguous
spectral editing with only two experiments, and conceivably, with only one scan each, e.g.,
for highly concentrated samples [11].
Experimentally, we found that with the DEPTQ experiment, the editing filter quality
in the Cq/CH and CH2/CH3 subspectra is not satisfactory, with a breakthrough of many
artifacts originating from other CHn multiplicities [13]. We also observed that for molecules
possessing large ranges of 1JCH coupling constants, DEPTQ135 spectra and edited subspec-
tra are obtained, with missing or very weak signals, especially CH3 or alkyne CH groups
(Figure 1). For instance, in the DEPTQ13590 spectrum of 4-methyl-N,N-di(prop-2-yn-1-
yl)aniline, the resonance of the methyl group C8 at 20.6 ppm is almost invisible (Figure 1).
While the DEPTQ9045 spectrum appears satisfactory, the edited CH2/CH3 subspectrum is
misleading, as the resonance of the methyl group C8 at 20.6 ppm is absent. We thought it
useful to develop a modified DEPTQ scheme for obtaining consistent DEPTQ and edited
subspectra for all classes of molecules. This could potentially be very attractive for rapid
13C analysis of newly synthesized or isolated compounds, especially for high-throughput
NMR analysis.
In this article, we present a new pulse sequence, DEPTQ+, possessing all these at-
tributes, and compare it with the modified DEPTQ experiment, designed to unequivocally
identify all carbon multiplicities (Cq, CH, CH2, and CH3) in two experiments. We show
both theoretically and experimentally that the DEPTQ+ pulse sequence always provides
unambiguous DEPTQ spectra with a leveling of all carbon intensities. This generally results
in processed spectra with a clean separation of the Cq and CH signals in one spectrum
and the CH2 and CH3 signals in the other, with both spectra showing the signals of the
two carbon species in opposite phases. It must be emphasized, however, that these proper-
ties only come into play and are relevant for molecules possessing a large range of 1JCH
coupling constants.

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Figure 1. 4-methyl-N,N-di(prop-2-yn-1-yl)aniline and carbon numbering and DEPTQ13590, DEPTQ9045, and the difference
between DEPTQ13590 and DEPTQ9045 spectra of ~30 mg of 4-methyl-N,N-di(prop-2-yn-1-yl)aniline dissolved in 0.7 mL
CDCl3. The delay
was set to 2.70 ms, adjusted for a coupling constant 1JCH of 185 Hz. The relaxation delay was 2 s, the
δ
NOE building period 1 s. All other parameters are identical to those described in the Materials and Methods section.
2. Results and Discussion
2.1. Theory
2.1.1. Original DEPTQ Pulse Sequence
Despite their known tolerance to the experimental parameters, edited DEPTQ spectra
may show undesirable residual CHn signals from “wrong” carbon types, a phenomenon
termed dubbed J cross-talk [21]. These signals arise, among other things, with non-ideally
1
adjusted delays and occur for both the coherences resulting from initial H- and from
13C-polarization. These signals are particularly troublesome in the pairwise edited CHn
spectra, as they can lead to misinterpretation, particularly if the editing is performed in an
automatic or semi-automatic way and with samples containing several, differently concen-
trated components [22
–27]. Therefore, this study predominantly focuses on suppressing
remaining CHn signals in the different subspectra as efficiently as possible.

Molecules 2021, 26, 3490
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1
The general condition δ = 1/(2 * JCH) is assumed in the following first product
operator treatments of the original DEPTQ pulse sequence (Figure 2). For simplicity, 1H-
and 13C-shift evolutions during the delays
δ
and the gradients G1–G3 used in practice are
not considered (the overall result is identical, except that only one of the 13C magnetization
terms at coherence level
1, C−, is detected if the PFGs are considered). We use here the
−
bracket notations proposed by Mateescu and Valeriu [28].
Figure 2. DEPTQ [11] (top) and DEPTQ+ pulse sequences (bottom) for editing CHn multiplicities.
◦
The first adjustable proton pulse
Ω
◦
and the editing pulse
θ
are set to 45 and 90o, respectively, for a
◦
DEPTQ90 (DEPTQ9045) and to 90 and 135 , respectively, for a DEPTQ135 (DEPTQ13590), and the
delay δ
is adjusted to 1/(2 * 1JCH). The DEPTQ9045 spectrum, showing exclusively the Cq and CH
signals with opposite phases, and the DEPTQ13590 spectrum, are subtracted from each other results in
a CH2/CH3 edited spectrum with the signals of the two carbon types with opposite phases [11]. The
◦
13C 180 (refocusing) pulses are composite smoothed chirp pulses (2 ms total duration, 60 kHz sweep
width). The initial 13C 90◦ pulse may be replaced by a variable
ϕ pulse, adjustable for maximum
sensitivity (Ernst angle). An additional 90◦ 13C pulse prior to data acquisition re-establishes the
residual 13C-z magnetization left with the initial 13C pulse. In the DEPTQ+, compared to the original
DEPTQ version [10
periods are defined as spin echoes, thus allowing different values for δ1
first adjustable proton pulse
DEPTQ+90 (DEPTQ+9045) and to 90 and 45o, respectively, for a DEPTQ+45 (DEPTQ+4590).
,
11], the first evolution delay
δ
is omitted, and the remaining three evolution
δ2, and δ3 to be used. The
Ω and the editing pulse θ
are set to 45◦ and 90◦, respectively, for a
,
◦
Quaternary Carbons
The first 13C pulse ϕ is set arbitrarily (Ernst angle)
◦
◦
◦
[z] ∅→yC cos∅[z] +sin∅[x] → cos∅[z] + sin∅[x] →
Ω xH18→0 xC→δ −cos∅[z]
δ
◦
→
◦
+sin∅[x] 180 xH90 xC
→ cos∅[y] + sin∅[x] → cos∅[y]
→ −cos∅[y] + sin∅[x] → → cos∅[z]
δ
(1)
θ◦yH
◦
δ 90◦−xC
+sin∅[x] 180 xC
+sin∅[x]
→
Of course, the outcome for quaternary carbons is identical in both experiments,
DEPTQ13590,
Ω Ω
= 90◦ and DEPTQ9045, = 45◦, irrespective of the length of the pro-
ton pulse θ.
DEPTQ135, Ω = 90◦, θ = 135◦

Molecules 2021, 26, 3490
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Subsequently, the following abbreviations will be used:
c = cos πJδ and s = sin πJδ c2 = cos 2πJCHδ and s2 = sin 2πJCHδ
For the observable signals originating from the polarization transfer from 1H to 13
C
(DEPT135), we obtain [7,8,29]:
1. CH groups
∅◦yC
(180◦)xH(90◦)xC
◦
◦
δ
δ
δ
[1z] → → [1z] →
→
90 xH180 xC −[1y] → s[zx]
→
→
−s[yx] →
◦
◦
−s[yx] (180 )xC
+0.7s[yz] → →
→
s[yx] 135 yH
→
0.7s[yx]
(2)
◦
1H−decoupling
δ 90 −xC
→
0.7s2[x1]
2. CH2 groups
∅◦yC
δ
δ 90◦−xC
[1z1] + [11z] → → . . . → → 0.35s22[x11] − s4[x11]
(3)
(4)
3. CH3 groups
∅◦yC
δ
δ 90◦−xC
[1z11] + [11z1] + [111z] → → . . . → → 0.53s22c2[x111]
−0.75s22s2[x111] + 1.06s6[x111]
The final carbon signal intensities, represented by I, for the three groups CH, CH2
and CH3 for and the actual
= 90◦ and = 135◦, as a function of the evolution delay
Ω
θ
δ
coupling constant JCH, are thus (the operators [x1], [x11], and [x111] have been replaced by
the Cartesian operator Cx for simplicity):
ICH = Cx [0.71 sin2(πJCHδ)]
ICH2 = Cx [0.35 sin2(2πJCHδ) − 1.00 sin4(πJCHδ)]
(5)
ICH3 = Cx [0.53 sin2(2πJCHδ) cos2(πJCHδ) − 0.75 sin2(2πJCHδ) sin2(πJCHδ) + 1.06
sin6(πJCHδ)]
which reduces with δ = 1/(2 * 1JCH) (matched delay) to:
ICH = +0.71 Cx
ICH2 = −1.00 Cx
ICH3 = +1.06 Cx
(6)
Equation (5) provides the analytical expressions to calculate the carbon signal in-
tensities for the three groups CH, CH2 and CH3 as a function of the evolution delay
δ
and the actual coupling constant JCH. Obviously, very weak resonances result in cases of
0
large mismatches between actual 1JCH coupling constants and the average 1JCH coupling
constant set (=1/(2 *
δ
)) in the DEPTQ experiment. In detail, large mismatches exist (i) for
acetylenic CH (1JCH ~ 250 Hz) or hetero aromatic rings (1JCH ~ 170–210 Hz) when 1JCH is
0
set to the classical value of 145 Hz, and (ii) for typical methyl groups (1JCH ~ 110–135 Hz)
1
0
1
when JCH is set to an average value 185 Hz, to account for the whole range of JCH
coupling constants (100–250 Hz) when acetylenic CH or hetero aromatic rings are present
(Figures S1 and S2).
DEPTQ90, Ω = 45◦, θ = 90◦
1. CH groups
∅◦yC
(180◦)xH(90◦)xC
◦
◦
δ
δ
[1z] → → →
δ 180 xC [1z] 45→xH −0.7[1y] → 0.7s[zx]
→
→
−0.7s[yx] →
(7)
90◦yH
◦
δ 90◦xC
−0.7s[yx] (180 )xC
→
0.7s[yx] → −0.7s[yz] → → 0.7s2[x1]

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2. CH2 groups
3. CH3 groups
∅◦yC
δ
δ 90◦−xC
[1z1] + [11z] → → . . . → → 0.35s22[x11]
(8)
(9)
∅◦yC
δ
δ 90◦−xC
[1z11] + [11z1] + [111z] → → . . . → → 2.11s2c4[x111]
The final carbon signal intensities, represented by I, for the three groups CH, CH2, and
◦
◦
CH3 for
Ω
= 45 and
θ
= 90 , as a function of the evolution delay
δ
and the actual coupling
JCH, are thus:
ICH = Cx [0.71 sin2(πJCHδ)]
ICH2 = Cx [0.35 sin2(2πJCHδ)]
(10)
ICH3 = Cx [2.11 sin2(πJCHδ) cos4(πJCHδ)]
which reduces with δ = 1/(2 * 1JCH) (matched delay) to:
ICH = +0.71 Cx
ICH2 = 0
(11)
ICH3 = 0
DEPTQ13590 − DEPTQ9045, edited CH2/CH3 subspectrum
The subtraction of the two data, DEPTQ13590 − DEPTQ9045, leads to:
ICH = Cx [0.71 sin2(πJCHδ)] − Cx [0.71 sin2(πJCHδ)] = 0
ICH2 = Cx [0.35 sin2(2πJCHδ) − 1.00 sin4(πJCHδ)] − Cx [0.35 sin2(2πJCHδ)] = −Cx
sin4(πJCHδ)
(12)
(13)
ICH3 = Cx [0.53 sin2(2πJCHδ) cos2(πJCHδ) − 0.75 sin2(2πJCHδ) sin2(πJCHδ) + 1.06
sin6(πJCHδ)] − Cx [2.11 sin2(πJCHδ) cos4(πJCHδ)]
which reduces with δ = 1/(2 * 1JCH) (matched delay) to:
ICH2 = −1.00 Cx
ICH3 = +1.06 Cx
For the typical J-coupling constant ranges (for which
δ is usually set to an average
value of 145 Hz), the artefacts originating from CH2 groups in the DEPTQ9045 spectrum
are expected to be weak: due to its sin2-dependence, the intensity of the cross-talk term
0.35 sin2(2
πJ
CHδ) in Equation (10) is generally small, which is also shown in Figure S3.
However, CH2 artifacts can also be very intense and actually troublesome when the full
J-coupling constant range is taken into account (100–250 Hz), for which is usually set
to a larger average value around 180 Hz (Figure S4) [19 30]. Likewise, for a CH3 group,
the intensity of the cross-talk term 2.11 sin2( CHδ) cos4(
CHδ) in Equation (10) and, thus,
δ
,
πJ
πJ
CH3 artifacts, will be very weak for the usual J-coupling constant ranges, as shown in
Figure S3, but can also be very intense and troublesome when the full J-coupling constant
range is considered (Figure S4) [19,30].
The DEPTQ experiment has two potential drawbacks: (i) the problem of missing or
very weak resonances in the DEPTQ13590 when large 1JCH coupling constant ranges are
considered, and (ii) the existence of artefacts, possibly strong, originating from CH2- and
CH3 groups in the DEPTQ9045 spectrum. We propose the modification of the DEPTQ
experience to remedy these two weaknesses.
2.1.2. The DEPTQ+ Pulse Sequence
The proposed DEPTQ+ pulse sequence is depicted in Figure 2 together with the
original DEPTQ pulse sequence [10]. Compared to the latter, the evolution periods of length

Molecules 2021, 26, 3490
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δ
= 1/(2 * 1JCH) are replaced by three spin echoes of different length [21
,30]. Interestingly,
these spin-echoes make it possible to omit the first evolution period in the standard DEPTQ
pulse sequence that must precede the DEPT pulse sequence for proper refocusing of the Cq-
magnetization generated with the first 13C excitation pulse. Therefore, as in the PENDANT
sequence [4,5
], both the 1H and 13C excitation pulses are synchronized. The total length of
DEPTQ+ is, thus, shorter than that of DEPTQ and identical to a standard DEPT experiment,
which will reduce the relaxation losses.
The advantages of the DEPTQ+ compared to the DEPTQ experiment, which result from
the individual tuning of the three spin echo periods to different 1JCH coupling constants
are: (i) a mutual leveling of the DEPT signal intensities, similar to that achieved with
the ACCORD-DEPT experiment, which systematically samples a range of 1JCH coupling
constants [31]. The accordion principle is, however, more complicated to implement than
tuning the three spin echo periods, as shown subsequently. (ii) A reduction in J cross-talk
in the CH/Cq spectrum (DEPTQ90), although this J cross-talk reduction is only noticeable
1
and disturbing for molecules with a large range of JCH coupling constants, as shown
theoretically (Figures S3 and S4), and subsequently in the experimental part.
Note that the intensities of quaternary carbon resonances are, of course, not affected
by these three echo periods used with DEPTQ+ and are the same as with DEPTQ. However,
compared to the DEPTQ experiment, note that while the first 13C pulse
ϕ is =
90◦, the
additional 180◦ pulses during the three spin echo periods invert the sign of the residual
z-magnetization that is not detected. Thus, the phase of the additional 90◦ 13C pulse prior
to data acquisition must be applied along the +x axis to re-establish the residual 13C-z
magnetization left with the initial 13C pulse.
Quaternary Carbons
The first 13C pulse φ is set arbitrarily (Ernst angle):
∅◦yC
◦
180◦xC
δ1
[z] → → →
−cos∅[z] + sin∅[x] 90→xC cos∅[y]
180 xC 180◦xC
◦
◦
δ2
δ
3
+sin∅[x] → → → → cos∅[y] + sin∅[x] 90→xC cos∅[z]
(14)
+sin∅[x]
The product operator evaluation for the CHn coherences originating from initial 1H-
polarization is applied in the DEPTQ+ pulse sequence, using the following abbreviations:
c = cos (πJCHδ1), s = sin (πJCHδ1)
c’ = cos (πJCHδ2), s’ = sin (πJCHδ2)
c00 = cos (πJCHδ3), s00 = sin (πJCHδ3)
s200 = sin (2πJCHδ3)
DEPTQ+13590, Ω = 90◦, θ = 135◦
The final carbon signal intensities, represented by I, for the three groups CH, CH2, and
CH3 for
constant JCH, are:
1. CH groups
Ω θ δ, and the actual coupling
= 90◦, = 135◦, as a function of the evolution delay
∅◦yC
◦
180◦xC180◦xH
90◦xC
δ1
[1z] 90→xH → → →
→
−[zx] . . . → −0.71ss00 [x1]
(15)
(16)
2. CH2 groups
◦
◦
◦
→
0
0
180◦xC180◦xH180◦xH
90◦xC
∅ yC
δ1
[1z1] + [11z] 90→xH90 xH
→ → →
→
→
. . . → 0.71sc0s200 [x11]
−ss0s002[x11]

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3. CH3 groups
[1z11] + [11z1]
◦
◦
◦
→
0
◦
→
◦
◦
◦
0
◦
◦
00
00
∅ yC
δ1
+[111z] 90→xH90 xH 90 xH
→ →18→0 xC180 xH180 xH 180 xH
→
→
→
00
3
. . . 90→xC
(17)
00
00
−2.11sc02s00 c [x111] + 3sc0s0s c00 [x111] − 1.06ss02s [x111]
2
2
The final carbon signal intensities, represented by I, for the three groups CH, CH2,
and CH3 for and the actual
= 90◦ and = 135◦, as a function of the evolution delay
Ω
θ
δ
coupling constant JCH, are thus:
ICH = Cx [−0.71 sin(πJCHδ1) sin(πJCHδ3)]
ICH2 = Cx [0.71 sin(πJCHδ1) cos(πJCHδ2) sin(2πJCHδ3) − 1.00 sin(πJCHδ1) sin(πJCHδ2)
sin2(πJCHδ3)]
ICH3 = Cx [−2.11 sin(πJCHδ1) cos2(πJCHδ2) sin(πJCHδ3) cos2(πJCHδ3) + 3 sin(πJCHδ1)
cos(πJCHδ2) sin(πJCHδ2) cos(πJCHδ3) sin2(πJCHδ3) − 1.06 sin(πJCHδ1) sin2(πJCHδ2)
sin3(πJCHδ3)]
(18)
DEPTQ+9045, Ω = 45◦, θ = 90◦
1. CH groups
∅◦yC
◦
→
◦
180◦xC180◦xH
◦
◦
180 xC
δ1
δ
2
[1z] 45→xH → → →
→
−0.71s[zx]
(90 )xC◦0.7s[yx] (180 )xH
→
→ →
◦
◦
δ 90◦xC
−0.71s[yx] 90→yH 0.71s[yz] (180 )xC(180 )xH
→
→
→ →
(19)
−0.71ss00 [x1]
2. CH2 groups
∅◦yC
◦
0
180◦xC180◦xH180◦xH
δ 90◦xC
δ1
[1z1] + [11z] 45→xH → → →
→
→
. . . → → −0.71sc0s200 [x11]
(20)
(21)
3. CH3 groups
∅◦yC
180◦xC180◦xH180◦xH
δ 90◦xC
◦
0
δ1
[1z11] + [11z1] + [111z] 45→xH → → →
→
→
. . . → →
00
2
−2.11sc02s00 c [x111]
The final carbon signal intensities, represented by I, for the three groups CH, CH2,
and CH3 for
Ω
= 45◦ and
θ
= 90◦, as a function of the three evolution delays δ1
,
δ2 and δ3
and the actual coupling JCH, are therefore:
ICH = −Cx [0.71 sin (πJCHδ1) sin (πJCHδ3)]
ICH2 = −Cx [0.71 sin (πJCHδ1) cos (πJCHδ2) sin (2πJCHδ3)]
(22)
ICH3 = −Cx [2.11 sin (πJCHδ1) cos2(πJCHδ2) sin (πJCHδ3) cos2(πJCHδ3)]
For the DEPTQ and DEPTQ+ experiments, the approximate carbon signal intensities,
◦
represented by I (for 1JCH
θ
≈
1JCH0), for the three groups CH, CH2, and CH3 for
Ω
= 90 and
= 135◦, as a function of the coupling constant 1JCH and
in Table 1:
δ, δ1, δ2, and δ3, are summarized
Table 1. Final carbon signal intensities, represented by I, for the three groups CH, CH2, and CH3 for
◦
◦
Ω = 90 , θ = 135 , as a function of the coupling constant JCH and the delays δ, δ1, δ2, and δ3.
DEPTQ
DEPTQ+
0.71Cxsin2(πJCHδ)
−Cxsin4(πJCHδ)
1.06Cxsin6(πJCHδ)
CH groups
CH2 groups
CH3 groups
−0.71Cxsin(πJCHδ1) sin(πJCHδ3)
−Cxsin(πJCHδ1) sin(πJCHδ2) sin2(πJCHδ3)
−1.06Cxsin(πJCHδ1) sin2(πJCHδ2) sin3(πJCHδ3)

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◦ ◦
Ω = 90 and θ = 135 and compared to the DEPTQ
According to the above relations for
experiment, the signs of the signal intensities for CH and CH3 groups are inverted using
the DEPTQ+ experiment, while the sign of the signal intensities for CH2 groups is identical.
Consequently, the usual angle
θ
for achieving the DEPT editing, CH/CH3 positive, CH2
negative, is obtained for
(Table 2).
θ
= 45◦ and not for = 135◦, as would be valid for DEPTQ
θ
Table 2. DEPTQ and DEPTQ+: maximal amplitude of CH, CH2, and CH3 groups for the three usual
◦
angles θ and for Ω = 90 .
Amplitude of
CH
Amplitude of
CH2
Amplitude of
CH3
Experiment
Angle θ
DEPTQ+
DEPTQ
DEPTQ+
DEPTQ
DEPTQ+
DEPTQ
−0.71
0.71
−1
1
1
−1.06
1.06
0
◦
45
0
◦
90
1
0
0
−0.71
0.71
−1
−1
−1.06
1.06
◦
135
To separate the signals of CH, Cq and CH2, CH3 groups in different sub-spectra and
to obtain different signs for the signals of CH2 and CH3 groups the first adjustable proton
pulse
Ω and the editing pulse θ
must therefore be set for the DEPTQ+ experiment to 45◦
and 90◦ (DEPTQ+9045) and to 90◦ and 45◦, respectively (DEPTQ+4590). Subtracting the
DEPTQ+9045 spectrum, showing exclusively the Cq and CH signals with opposite phases,
from the DEPTQ+4590 spectrum results in a CH2/CH3 edited spectrum with the signals of
the two carbon types with opposite phases.
Interestingly, equations 15–22 show that the intensities of the signals of the three carbon
types depend differently and for CH solely on two (δ1 and δ3) of the individual delays δ1, δ2,
and δ3, which is the basis for efficiently leveling the signal intensities for all multiplicities
with the DEPTQ+ pulse sequence, even for large 1JCH coupling constant ranges. Indeed,
and with the specific ranges of 1JCH-coupling constants for the three carbon types, i.e., CH:
[120 < 1JCH < 250 Hz], CH2: [115 < 1JCH < 175 Hz], CH3: [100 < 1JCH < 135 Hz], the three
delays δ1, δ2, and δ3 can be individually and optimally adjusted. For instance, δ2 which does
not affect CH-intensities can be adjusted for small 1JCH coupling constants, typically 120 Hz,
to maximize the signals of CH2 and CH3 groups. On the other hand, δ1 can be adjusted for
large 1JCH coupling constants, typically 220–230 Hz, to maximize the signals of acetylenic
CH groups. Finally, δ3 can be adjusted for an average 1JCH coupling constant, typically
185 Hz, to account for all carbon types present. This enables real leveling of the signal
intensities with the DEPTQ+ experiment, as demonstrated theoretically (Figures S3–S5)
and, in the following section, practically.
,
2.2. Experimental Results
2.2.1. Cholesteryl Acetate
We first used a sample of 100 mmol cholesteryl acetate (Figure 3, 30 mg dissolved in
0.7 mL CDCl3) for testing the performance of the DEPTQ+ experiment.

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Figure 3. Cholesteryl acetate and carbon numbering.
In Figures 4 and 5, the DEPTQ13590, DEPTQ9045, the difference between DEPTQ13590
and DEPTQ9045 (Figure 4), the DEPTQ+4590, DEPTQ+9045, and the difference between
DEPTQ+4590 and DEPTQ+9045 (Figure 5) of cholesteryl acetate are shown. Cholesteryl
1
acetate (Figure 3) is a molecule exhibiting a narrow range of coupling constants JCH
(122 Hz for 1JC26H26 to 155 Hz for 1JC6H6). Accordingly, both the DEPTQ9045 providing
a Cq/CH edited spectrum and the DEPTQ9045 providing a CH2/CH3 edited spectrum
(difference between DEPTQ13590 and DEPTQ9045) are of high quality, with few and weak
CH2/CH3 artefacts in the DEPTQ9045 and some residual CH peaks in the difference
spectrum, respectively (Figure 4).
Figure 4. Bottom: DEPTQ13590; middle: DEPTQ9045; top: Difference between DEPTQ13590 and DEPTQ9045 spectra of
30 mg of cholesteryl acetate dissolved in 0.7 mL CDCl3. The delay δ was set to 3.45 ms, adjusted for a coupling constant
1JCH of 145 Hz.

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Figure 5. Bottom: DEPTQ+4590
;
middle: DEPTQ+9045
;
top: Difference between DEPTQ+4590 and
DEPTQ+9045 spectra of 30 mg of cholesteryl acetate dissolved in 0.7 mL CDCl3. The delays δ1
,
δ2
,
and δ3 were set to 3.13 ms, adjusted for a coupling constant 1JCH of 160 Hz, to 4.35 ms, adjusted
1
1
for a coupling constant JCH of 115 Hz, and to 3.45 ms, adjusted for a coupling constant JCH of
145 Hz, respectively.
The same, nearly identical results have been obtained using the proposed DEPTQ+
pulse sequence. Thus, the DEPTQ+ behaves similarly to the DEPTQ experiment if the
1
narrow, usual range of coupling constants JCH is present: the mutual signal intensity
leveling and the J cross-talk reduction only provide very minor improvements.
However, the superiority of the DEPTQ+ experiment becomes clear when there is a
larger range of coupling constants 1JCH. First, with the sole aim of demonstrating the poten-
tial and performance of the DEPTQ+ experiment, we used the same molecule of cholesteryl
acetate but adjusted the three delays in the experiments as if all 1JCH coupling constants
(alkanes, aromatics, alkynes) were present. The spectra are shown in Figures S8 and S9.
In this case, it is clear that the DEPTQ+ behaves much better than the DEPTQ ex-
periment; even the standard DEPTQ135 spectrum is confusing, since the resonances of
the methyl groups between 10 and 30 ppm are barely visible (Figure S8), as predicted
theoretically (Figure S2). This is due to the very large mismatch between the actual 1JCH
coupling constants in methyl groups (~125 Hz) and the average 1JCH0 coupling constant
set in the DEPTQ experiment, 1JCH0 = 185 Hz, intentionally set to cover the whole range
of 1JCH coupling constants (100–250 Hz). The DEPTQ9045 is also confusing, with many
residual CH2/CH3 signals present, which makes the identification of CH groups diffi-
cult, as they are as intense (strong J cross talk) and have the same sign as the CH signals
(Figure S8). Finally, the “CH2/CH3” edited spectrum (difference between DEPTQ13590
and DEPTQ9045) is extremely confusing, as only the resonances of CH2 groups are present,
while the resonances of CH3 groups are either barely visible or even completely absent
(Figure S8).
In contrast, the spectra obtained using the DEPTQ+ pulse sequence are much better
and sufficient for reliable identification of CHn groups: In the DEPTQ+4590 spectrum, the
resonances of the methyl groups between 10 and 30 ppm are clearly visible and intense
(Figure S9), as predicted theoretically (Figure S7). This is because the three delays δ1
,
δ2
,
0
and δ3 in the DEPTQ+ experiment are matched to three different 1JCH coupling constants
(230, 125, 165 Hz, respectively), which results in a mutual leveling of the DEPT signal

Molecules 2021, 26, 3490
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intensities. In the DEPTQ+9045, there are only very few and weak CH2/CH3 artefacts
(weak J cross talk), which enables easy identification of CH groups (Figure S9). Finally,
the “CH2/CH3” edited spectrum also allows a reliable identification of the clearly visible
resonances of CH2 groups and of CH3 (Figure S9).
2.2.2. 4-Methyl-N,N-di(prop-2-yn-1-yl)aniline
In order to test the effective performance, we finally tried the DEPTQ+ experiment on
the demanding sample of ~30 mg of 4-methyl-N,N-di(prop-2-yn-1-yl)aniline dissolved in
0.7 mL CDCl3 (Figure 1). 4-methyl-N,N-di(prop-2-yn-1-yl)aniline contains alkane, aromatic
and alkyne groups with the full range of 1JCH coupling constants (125 Hz for 1JC8H8 to
1
248 Hz for JC1H1). Note also the very particular case of C2 at 79.4 ppm, which, with
its large long-range coupling (2JC2H1 ~ 50 Hz), behaves like a pseudo- or only partially
quaternary carbon.
In Figures 6 and 7, the DEPTQ13590, DEPTQ9045, the difference between DEPTQ13590
and DEPTQ9045 (Figure 6), and the DEPTQ+4590, DEPTQ+9045, and the difference be-
tween DEPTQ+4590 and DEPTQ+9045 (Figure 7) of 4-methyl-N,N-di(prop-2-yn-1-yl)aniline
are shown.
Figure 6. Bottom: DEPTQ13590
~30 mg of 4-methyl-N,N-di(prop-2-yn-1-yl)aniline dissolved in 0.7 mL CDCl3. The delay
,
middle: DEPTQ9045
;
top: Difference between DEPTQ13590 and DEPTQ9045 spectra of
was set to 2.70 ms, adjusted for
δ
a coupling constant 1JCH of 185 Hz. The relaxation delay was 4 s, the NOE building period 3 s. All other parameters are
identical to those described in the Materials and Methods section. The structure of 4-methyl-N,N-di(prop-2-yn-1-yl)aniline
and carbon numbering is shown below the spectra.

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Figure 7. Bottom: DEPTQ+4590
of ~30 mg of 4-methyl-N,N-di(prop-2-yn-1-yl)aniline dissolved in 0.7 mL CDCl3. The delays δ1
;
middle: DEPTQ+9045
;
top: Difference between DEPTQ+4590 and DEPTQ+9045 spectra
δ2, and δ3 were set to
,
2.18 ms, adjusted for a coupling constant 1JCH of 230 Hz, to 4.00 ms, adjusted for a coupling constant 1JCH of 125 Hz, and
to 3.18 ms, adjusted for a coupling constant 1JCH of 165 Hz, respectively. The relaxation delay was 4 s, the NOE building
period 3s. All other parameters are identical to those described in the Materials and Methods section. The structure of
4-methyl-N,N-di(prop-2-yn-1-yl)aniline and carbon numbering is shown below the spectra.
With 4-methyl-N,N-di(prop-2-yn-1-yl)aniline, it is clear that the DEPTQ+ performs
much better than the DEPTQ experiment: the standard DEPTQ135 spectrum is useable,
but the resonance of the methyl group C8 at 20.6 ppm is very weak (Figure 6). As already
1
mentioned, this is due to the very large mismatch between the actual JCH coupling
0
constants in methyl groups (~125 Hz) and the large average 1JCH coupling constant set in
the DEPTQ experiment, 1JCH0 = 185 Hz (Figure S2). Artefacts (J cross talk) belonging to the
CH2 C3 at 40.9 ppm and to the CH3 C8 at 20.6 ppm are visible in the DEPTQ9045, even if
their intensities are rather small (Figure 6). Such artefacts can be problematic with unknown
molecular structures or if the attribution is performed in an automatic or semi-automatic
way using dedicated attribution software. Particularly confusing is the “CH2/CH3” edited
spectrum obtained with DEPTQ (difference between DEPTQ13590 and DEPTQ9045), with a
strong signal of the CH2 groups C3 at 40.9 ppm and a weak residual signal of the CH group
C1, while the resonance of the CH3 group C8 at 20.6 ppm is almost invisible (Figure 6). In
addition, the residual signal of C1 at 72.8 ppm could be misleading or misinterpreted by
attribution/analysis software.

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In contrast, the spectra obtained using the DEPTQ+ pulse sequence are less confusing
and sufficient for reliable carbon type identification: in the DEPTQ+4590, the resonance of
the methyl group C8 around 20 ppm is clearly visible and intense (Figure 7). This must be
0
attributed to the individual tuning of the three delays to different 1JCH coupling constants
(125, 175, 230 Hz), which results in a mutual leveling of the DEPT signal intensities. The
DEPTQ+9045 shows—besides the negative quaternary carbon signals—the three strong CH
signals with only one weak CH2 artifact (weak J cross talk) present (Figure 7). Finally, the
CH2/CH3 edited spectrum is almost perfect, with the strong resonances of the CH2- and
CH3 groups C3 and C8 in anti-phase and only very weak residual signals left (Figure 7).
2.3. Practical Aspects
Our theoretical and practical investigations show that the proposed new DEPTQ+4590
and DEPTQ+9045 experiments work well. However, the experimental results suggest that
the quality of spectral editing also depends on the relaxation delay. While for medium sized
molecules such as cholesteryl acetate, the length of the relaxation delay affects only the
intensity of the slow relaxing carbons but not the editing quality (Figure S10), it becomes
critical also for the editing procedure for small molecules and/or groups exhibiting long T1
relaxation times.
This manifests itself with 4-methyl-N,N-di(prop-2-yn-1-yl)aniline, for which the in-
tensities of the CH signals in the DEPTQ+9045 spectra can be drastically different to that
obtained in the DEPTQ+4590 if overly short relaxation delays are used. The numerous
and intense “CH-artefacts” showing up in the edited CH2/CH3 spectrum may be poten-
tially attributed to CH3 groups. The influence of relaxation and the need for choosing the
relaxation delay(s) long enough with DETPQ+ to achieve a sufficient editing quality are
corroborated by simulated spectra, which are in good agreement with the corresponding
experimental spectra shown in Figures S11 and S12.
Finally, we wish to concentrate on the overall sensitivities of DEPTQ+, and to com-
pare it with DEPTQ and the usually performed “tandem alternatives” such as 13C one-
pulse/DEPT. In previous reports [9–11], Bigler et al. have compared spectra acquired
DEPTQ and the 13C one-pulse spectra with an equal number of scans within the same
total measuring time, and found that the DEPTQ spectrum decreases signal intensities
of quaternary carbons by 15% and 10% when compared to the 13C one-pulse. The corre-
sponding experiments (DEPTQ vs. 13C one-pulse + DEPT) based on equal total measuring
times showed average S/N ratios of about 70% and 85% for the CHn signals in the DEPT
spectrum, compared to the corresponding values of the DEPTQ experiment [10]. While
the signal intensities of quaternary carbons are identical in DEPTQ and DEPTQ+ spec-
tra, the comparison of the average S/N ratio for the CHn signals between DEPTQ and
DEPTQ+ is not so straightforward. While for standard DEPTQ experiments, as shown in
this manuscript, the signals at the extremes of the 1JCH coupling range suffer in intensity
or cannot be detected, DEPTQ+ experiments (similar to ACCORD-DEPT experiments [31])
provide spectra with notably improved S/N ratios for these specific signals. However,
owing to the mutual signal intensity leveling, DEPTQ+ always provides either better or
worse signal intensity compared to standard optimization. The S/N ratio for some signals
will correspondingly decrease compared to a standard optimized DEPTQ experiment.
3. Materials and Methods
3.1. Synthesis and Characterization of 4-Methyl-N,N-di(prop-2-yn-1-yl)aniline
The chemicals were purchased from Aldrich, Alfa Aesar, Acros Organics, and TCI
Chemicals and used without further purification, In a flame-dried round bottom flask
equipped with a magnetic stirrer, p-toluidine (0.5 g, 4.6 mmol) and anhydrous K2CO3
(0.77 g, 5.52 mmol) were mixed in dry dimethylformamide (10 mL). Propargyl bromide
(0.65 g, 5.52 mmol) was then added dropwise during a period of 15 min and the resulting
mixture was stirred under nitrogen atmosphere for 5 h. After completion of the reaction
(monitored by TLC), the mixture was poured into water (10 mL) and extracted with ethyl

Molecules 2021, 26, 3490
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acetate (2
×
15 mL). The combined organic layers were washed with water (4
×
10 mL)
and brine (10 mL), dried over sodium sulfate, filtered, and concentrated under reduced
pressure [32 33]. The reaction crude was purified by column chromatography (Silica,
,
230–400 mesh) using mixtures of hexane/ethyl acetate (30:1). The desired product was
obtained as a highly dense yellow oil (110 mg, 13%) along with the product of mono
substitution 4-methyl-N-(prop-2-yn-1-yl)aniline (500 mg, 75%).
1
4-methyl-N,N-di(prop-2-yn-1-yl)aniline: H NMR (300 MHz, CDCl3)
δ
7.12 (d, J = 8.6 Hz,
2H), 6.92 (d, J = 8.6 Hz, 2H), 4.11 (d, J = 2.4 Hz, 4H), 2.30 (s, 3H), 2.26 (t, J = 2.4 Hz, 2H).
13C NMR (75 MHz, CDCl3)
δ
145.8 (C4), 129.8 (C5), 129.7 (C7), 116.7 (C6), 79.4 (C2), 72.8
(C1), 40.9 (C3), 20.6 (C8). The spectra are referenced with respect to the residual CHCl3
resonance (7.264 ppm) and to the 13C resonance of CDCl3 (77.16 ppm).
3.2. NMR Measurements
All DEPTQ and DEPTQ+ experiments were recorded on a Bruker AvanceII 500 MHz
NMR spectrometer equipped with a 5 mm BBI Inverse probehead. The samples used
were: (i) the standard test sample provided with Bruker NMR spectrometers, 30 mg of
cholesteryl acetate dissolved in 0.7 mL CDCl3, and (ii) ~30 mg of 4-methyl-N,N-di(prop-2-
1
yn-1-yl)aniline dissolved in 0.7 mL CDCl3. H and 13C 90◦ pulse lengths were 8.4
µs and
13.2 µs, respectively. The composite chirp pulse for refocusing has a duration of 2 ms, is
defined by 4000 points and by 20% smoothing and 60 kHz sweep width (Crp60comp.4 in
the Bruker wave form library). The experimental details of the measurements are as follows.
Cholesteryl acetate: if not otherwise mentioned, all spectra were acquired with 128 k real
data points with a relaxation delay of 4 s, a NOE building period of 3 s, 64 scans for a total
experimental time of approximately 7 min. 13C spectral widths were 200 ppm, leading to
an acquisition time of 2.60 s. Data were processed with 128 k data points using exponential
multiplication with a line broadening of 1 Hz. 4-methyl-N,N-di(prop-2-yn-1-yl)aniline:
the spectra were acquired with 128 k data points with a relaxation delay of 10 s, a NOE
building period of 5 s, 16 scans for a total experimental time of approximately 3 min. 13C
spectral widths were 200 ppm, leading to an acquisition time of 2.60 s. Data were processed
with 128 k data points using exponential multiplication with a line broadening of 1 Hz.
3.3. Numerical Simulations
The numerical simulations shown in Figures S1–S7 have been performed with Mi-
crosoft Excel®, Office 365 for Windows. Relaxation effects during both pulse sequences
were not considered.
3.4. NMR Simulations
The simulations shown in Figures S10 and S11 have been performed with the BRUKER
NMRSIM program for MAC (version 5.5.3. 2012). The simulated DEPTQ and DEPTQ+
spectra are obtained for 4-methyl-N,N-di(prop-2-yn-1-yl)aniline. The parameters used
1
1
1
1
for the simulations were: JC1H1 = 260 Hz, JC3H3 = 145 Hz, JC5H5 = JC6H6 = 165 Hz,
1JC8H8 = 125 Hz, 2JC2H1 = 50 Hz. The relaxation times have been set to T1 = 1 s for all CHn
groups, and T1 = 5 s for the quaternary carbons, T2 = 0.5 s, and full relaxation during both
the pulse sequence and final data acquisition were considered. The chemical shifts set in
the simulations were the experimental chemical shifts.
4. Conclusions
Compared to the DEPTQ experiment, the proposed new DEPTQ+ experiment is
shorter and the different evolution delays are designed as spin echoes, which can be
matched to different 1JCH values. We have shown, both theoretically and experimentally,
that for molecules with a standard range of 1JCH coupling constants (i.e., 110,180 Hz), the
differences between DEPTQ and DEPTQ+ are minimal. The differences become appreciable
for molecules possessing a large range of 1JCH coupling constants. The DEPTQ+ experiment
makes it possible to obtain DEPTQ spectra with (i) a mutual leveling of the DEPT signal

Molecules 2021, 26, 3490
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intensities, (ii) a reduction in J cross-talk in the Cq/CH spectrum, and (iii) more reliable
CH2/CH3 edited spectra.
The influence of relaxation and the need to choose a relaxation delay that is sufficiently
long, with DETPQ+, to obtain reliable data and clean edited spectra represents the only
limitation of this study, but this is valid for molecules with long T1 relaxation times. This is,
however, also the case with the original DEPTQ experiment.
In conclusion, the new DEPTQ+ experiment is expected to be attractive for fast 13
C
analysis of small-to medium sized molecules, especially those with a large range of 1JCH
coupling constant, and especially in high-throughput laboratories. With concentrated sam-
ples and/or by exploiting the high sensitivity of cryogenically cooled 13C NMR probeheads,
the efficacy of such investigations may be improved, as it is possible to unequivocally
identify all carbon multiplicities with only one scan for each of the two independent
DEPTQ+ experiments.
Supplementary Materials: The following are available online. Figure S1: DEPTQ13590: theoretical
amplitude of a CH group as a function of the 1JCH coupling constant, Figure S2: DEPTQ13590: theo-
retical amplitude of CH3 groups as a function of the 1JCH coupling constant, Figure S3: DEPTQ9045
1
and DEPTQ+9045: theoretical residual amplitude of CH2 and CH3 groups, usual JCH coupling
constant range, Figure S4: DEPTQ9045 and DEPTQ+9045: theoretical residual amplitude of CH2 and
CH3 groups, full 1JCH coupling constant range, Figure S5: DEPTQ9045 and DEPTQ+9045: theoretical
amplitude of CH groups, full 1JCH coupling constant range, Figure S6: DEPTQ9045 and DEPTQ+9045
:
theoretical amplitude of CH2 groups, full 1JCH coupling constant range, Figure S7: DEPTQ9045 and
DEPTQ+9045: theoretical amplitude of CH3 groups, full 1JCH coupling constant range, Figure S8:
DEPTQ13590, DEPTQ9045 and (CH2/CH3) edited spectra of cholesteryl acetate, adjusted for a cou-
pling constant 1JCH of 185 Hz, Figure S9: DEPTQ+4590, DEPTQ+9045 and (CH2/CH3) edited spectra
of cholesteryl acetate, Figure S10: DEPTQ and DEPTQ+ spectra of cholesteryl acetate: influence of the
relaxation delay, Figure S11: Theoretical and DEPTQ and DEPTQ+ spectra of 4-methyl-N,N-di(prop-
2-yn-1-yl)aniline: relaxation delay = 2s, Figure S12: Theoretical and DEPTQ and DEPTQ+ spectra of
4-methyl-N,N-di(prop-2-yn-1-yl)aniline: relaxation delay = 20s.
Author Contributions: Conceptualization, P.B. and J.F.; methodology, P.B., C.M. and J.F.; software,
P.B. and J.F.; validation, P.B. and J.F.; formal analysis, P.B. and J.F.; investigation, P.B. and J.F.;
resources, J.F.; data curation, P.B. and J.F.; writing—original draft preparation, P.B., C.M. and J.F.;
writing—review and editing, P.B. and J.F.; visualization, P.B. and J.F.; supervision, P.B. and J.F.; project
administration, J.F.; funding acquisition, J.F. All authors have read and agreed to the published
version of the manuscript.
Funding: This research and the APC were funded by the University of Berne.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compound 4-methyl-N,N-di(prop-2-yn-1-yl)aniline are available
from the authors. Cholesteryl acetate is commercially available.
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