Catalysis of Hydrogen-Deuterium Exchange Reactions by 4-Substituted Proline Derivatives

Article abstract of DOI:10.1021/acs.joc.8b02644

The identification and understanding of structure-activity relationships is vital for rational catalyst design. A kinetic study of the hydrogen-deuterium exchange reaction of cyclohexanone in aqueous solution, as catalyzed by proline derivatives, has revealed valuable structure-activity relationships. In phosphate-buffered solution, cis-4-fluoroproline is more active than the trans isomer, a distinction that appears to originate from a destabilizing interaction between the fluorine atom and phosphate anion during general acid-catalyzed dehydration of the carbinolamine intermediate. trans-4-Ammoniumprolines are exceptionally active catalysts owing to favorable Coulombic interactions involving the ammonium group and the alkoxide moiety formed upon 1,2-addition of the proline derivative to the ketone. These results could be used for the optimization of proline catalysts, especially in transformations where the formation of the putative iminium ion is rate-limiting.

Full text of DOI:10.1021/acs.joc.8b02644

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Article
Catalysis of Hydrogen–Deuterium Exchange
Reactions by 4-Substituted Proline Derivatives
Eddie L. Myers, Michael J Palte, and Ronald T Raines
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02644 • Publication Date (Web): 02 Jan 2019
Downloaded from http://pubs.acs.org on January 3, 2019
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Catalysis of Hydrogen–Deuterium Exchange Reactions by
4-Substituted Proline Derivatives
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Eddie L. Myers,*,†,‡ Michael J. Palte,§ and Ronald T. Raines*,#,
⊥
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†Department of Biochemistry, University of Wisconsin–Madison, 433 Babcock Drive,
Madison, Wisconsin 53706, United States. ‡School of Chemistry, NUI Galway, University
Road, Galway, Ireland. §Medical Scientist/Molecular and Cellular Pharmacology Training
Programs, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States.
#Department of Chemistry, University of Wisconsin–Madison, 1101 University Avenue,
Madison, Wisconsin 53706, United States. ⊥Department of Chemistry, Massachusetts
Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United
States.
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ABSTRACT: The identification and understanding of structure–activity relationships is vital
for rational catalyst design. A kinetic study of the hydrogen–deuterium exchange reaction of
cyclohexanone in aqueous solution, as catalyzed by proline derivatives, has revealed valuable
structure–activity relationships. In phosphate-buffered solution, cis-4-fluoroproline is more
active than the trans isomer, a distinction that appears to originate from a destabilizing
interaction between the fluorine atom and phosphate anion during general acid catalyzed
dehydration of the carbinolamine intermediate. Trans-4-ammoniumprolines are exceptionally
active catalysts owing to favourable Coulombic interactions involving the ammonium group
and the alkoxide moiety formed upon 1,2-addition of the proline derivative to the ketone. These
results could be used for the optimisation of proline catalysts, especially in transformations
where the formation of the putative iminium ion is rate-limiting.
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INTRODUCTION
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Transformations of saturated and unsaturated aldehydes and ketones in reactions catalyzed by
small-molecule secondary amines, such as proline, are prevalent in organic synthesis.1
Although initial reports on such aminocatalysis began as early as the late 19th century,2 it was
the appearance of reports from the research groups of Barbas, Lerner, List, and MacMillan in
the late 1990s that began sustained interest in this area.3 Since the appearance of these early
reports, aminocatalytic reactions have been shown to transform readily available substrates into
diverse products with high levels of asymmetric induction, though turnover numbers remain
low.4 The activity of small-molecule organocatalysts in water, in particular, is of interest
because of potential benefits for the development of both environmentally friendly synthetic
processes5 and tools for chemical biology.6 Furthermore, these investigations—and the
exploration of the activity of enzymes in organic solvents—7 addresses the discontinuity
between modern organocatalysis and native enzyme catalysis. The field of aminocatalysis is
now maturing in that studies toward gaining greater mechanistic insight are taking center
stage,8 a development that should facilitate catalyst design and could lead to commercially
viable aminocatalytic processes.9
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With an appreciation of the effect of 4-substituted prolines on the structure of protein,10
we decided to investigate the catalytic activity of 4-substituted prolines on a very simple
transformation—the hydrogen–deuterium exchange reaction of ketones in aqueous solution,
one of the earliest transformations to be investigated in the context of small-molecule
aminocatalysis.11 In a preliminary experiment (Scheme 1), we found that the catalytic activity
of fluoroprolines on the hydrogen–deuterium exchange reaction of cyclohexanone in
phosphate-buffered D2O (pD 7.4) increased in the order trans-4-fluoroproline (3, pKa 9.7) <
cis-4-fluoroproline (2, pKa 9.7) << 4,4-difluoroproline (1, pKa 7.8).12 The higher activity of cis-
4-fluoroproline relative to the trans isomer (>2 fold) was intriguing and warranted further
investigation. The relative levels of activity of 4-fluoroprolines has been investigated
previously in the context of other organocatalytic reactions in organic solvents.13 For example,
in a transannular aldol reaction, the use of the trans isomer gave higher yields and higher levels
of asymmetric induction than that of the cis isomer. 13b The different levels of activity were
proposed to originate from the strong preference of 4-fluoroproline derivatives to adopt a
pyrrolidine ring pucker that places the fluoro group in a pseudo-axial orientation, a preference
that has been attributed to the gauche effect.14 To determine the origin of the different levels of
activity exhibited by the fluoroprolines we needed to fully understand the mechanism of the
proline-catalyzed hydrogen–deuterium exchange reaction of cyclohexanone. In the 1960s and
1970s, Hine and co-workers found that the identity of the rate-limiting intermediate/transition
state in amine-catalyzed isotope-exchange reactions of aldehydes and ketones depends on the
nature of the catalyst, the substrate, and the reaction conditions.15 However, the exchange
reaction has not been investigated for cyclohexanone and proline-based amines, leaving
insufficient data to deduce the origin of the different levels of activity exhibited by the 4-
fluoroproline derivatives. Herein, we show that the identity and orientation of substituents in
4-substituted proline derivatives have a substantial effect on the activity of these derivatives in
the exchange reaction, and we explain the origin of these effects through the analysis of reaction
kinetics.
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O
O
fluoroproline
150 mM phosphate
(10 mol%)
Dn
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7
8
D2O, pD 7.4 (I=0.9), RT
n = 04
[D]-A
[D]-B
[D]-C
[D]-D
F
F
F
F
<
<
CO2
CO2
CO2
N
N
N
9
D
D
D
D
D
D
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3
1
(pKa 7.8)
(pKa 9.7)
2
(pKa 9.7)
Rel. rate
1
2.4
7.6
Y
Y
X
X
Y
Y
Y
CO2
CO2
OD
CO2
X
X
X
CO2
CO2
N
N
N
N
N
O
OD
D
D
C
A
B
D
E
Scheme 1: Relative catalytic activity of fluoroprolines at pD 7.4 and putative intermediates in
the isotope exchange reaction of cyclohexanone.
Results and Discussion:
The rate of incorporation of deuterium into the α positions of cyclohexanone in
phosphate-buffered D2O at various pD values, as catalyzed by fluoroprolines 1–3, was
measured by 1H NMR spectroscopy up until 10% conversion (Figure 1A). Spectra showed sets
of resonances for the proline derivative and [D0]-cyclohexanone and its isotopologues;
resonances associated with intermediates were not detected. The pD–rate profiles exhibit a
maximum at pD values close to the pKa value of the zwitterionic proline derivative. The
exchange reaction was fastest at pD values close to 7 in the presence of 4,4-difluoroproline (1;
pKa 7.8), and cis-4-fluoroproline 2 (pKa 9.7) is an approximate 2-fold more active catalyst than
trans-4-fluoroproline 3 (pKa 9.7). Catalysis by fluoroprolines 1–3 is much more efficient than
by unmodified proline: at ~pD 7.4, the rate of exchange as catalysed by difluoroproline 1 is
approximately 100 times that for proline. Above pD 10.0, as the pKa value of proline is
approached, the rate of exchange as catalyzed by proline increases rapidly; however, at these
pD values, the background rate (buffer catalysis) also becomes significant (see Supporting
Information). For each proline derivative, the order of the reaction with respect to each reaction
component was determined using log/log plots of data obtained from the measurement of
reaction rates at various concentrations of the reaction component, while keeping all other
variables constant. The reactions exhibit component-specific orders of ~1 for cyclohexanone,
the proline derivative, and phosphate at pD values close to maximum rate. The reaction order
of phosphate buffer was determined by subtracting the low background rate associated with
the aminocatalyst acting as both the putative covalent catalyst and as a donor/acceptor of
deuteron, thus the value of ~1 only includes transformations mediated through putative
covalent catalysis by the proline derivative. Owing to the first order dependence of the rate of
exchange on the buffer species, data for the reactions in imidazole-buffered D2O were also
obtained (Figure 1B). Interestingly, under these conditions, the cis- and trans 4-fluoroprolines
exhibited similar levels of activity, suggesting that the different levels of activity in phosphate
buffer originate from an intimate interaction between a phosphate species and a fluoroproline-
containing intermediate.
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Figure 1. (A) pD–rate profiles of exchange reactions catalyzed by 1–3 in phosphate-buffered
D2O (I = 0.9); (B) pD–rate profiles of exchange reactions catalyzed by 2 and 3 in imidazole-
buffered D2O (I = 0.15); (C) pD–rate profiles of the exchange reaction catalyzed by 1 in
D2O/H2O (1:1, I = 0.9); (D) Rate of exchange reactions of [D0]- and [D4]cyclohexanone,
catalyzed by 1 in D2O/H2O (1:1, pH-meter reading 7.2, I = 0.9).
Substrate isotope effects were determined by measuring the rates of exchange of the α
hydrogen atoms of [D0]-cyclohexanone and the α deuterium atoms of [D4]-cyclohexanone as
catalyzed by 1 in 1:1 mixtures of phosphate-buffered D2O and H2O (150 mm, I = 0.9). The use
of a common solvent (H2O/D2O, 1:1) allows the substrate isotope effect to be disentangled
from solvent isotope effects. GC-MS analysis of aliquots taken from reaction mixtures at pH-
meter values of 6 and 8 with consideration of the fractionation factors (φ, [DA]/[HA] in
D2O/H2O 1:1) of the major deuteron- and proton-donating species at these pH-meter values
2–
2−
(D2PO4–/H2PO4–, φ = 1.03; DPO4 /HPO4 , φ = 0.91),16 gave kH/kD values of ~1 (Figure 1D,
Table 1). The solvent isotope effect for the exchange reaction catalyzed by proline 1 was
determined by comparing rates of reactions carried out in D2O with those in D2O/H2O (1:1) at
various pH-meter values (Figure 1C). Considering that approximately half of the exchange
events in D2O/H2O (1:1) are unobservable, that is, a hydrogen atom is exchanged for another
hydrogen atom (as supported by substrate isotope effects), the maximum rate of exchange in
D2O was found to be ~5-fold higher than that in D2O/H2O (1:1), thus pointing toward a
remarkably large inverse solvent isotope effect.
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Table 1. Substrate Isotope Effects Determined in H2O/D2O (1:1).
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pH meter reading [H2O]/[D2O] [DA]/[HA] kH→D/kD→H kH/kD
6.0
8.0
1.0
1.0
1.03
0.91
1.02
0.92
0.99
1.01
The absence of a primary isotope effect is consistent with the rate-determining step
occurring prior to the putative interconversion of iminium D and enamine E (Scheme 1). The
absence of a significant secondary isotope effect, which, for example, can range from 0.8–1.2
for addition reactions of [D0]- and [D6]-acetone,17 is consistent with one of two scenarios: i)
proline 1 undergoes rate-determining addition to cyclohexanone with an early transition state;
ii) the putative carbinolamine C, which is in equilibrium with starting material, undergoes rate-
determining dehydration, the associated kinetic secondary isotope effect completely offsetting
the preceding equilibrium secondary isotope effect. Additionally, analysis of the change in the
relative amounts of [D0]-cyclohexanone and its isotopologues over the course of the reaction
show that only one deuterium atom is incorporated at a time, that is, [D2]-cyclohexanone is
formed via [D1]-cyclohexanone (see mass spectrometry data in the Supporting Information).
This observation suggests that the distal and proximal axially orientated α hydrogen atoms with
respect to the carboxylate group of the putative iminium ion undergo abstraction as protons
with very different rates and that [D1]-iminium ion ([D1]-D1, Scheme 2) is converted into [D1]-
cyclohexanone faster than any process that would lead to its conversion into a
stereoisotopomer, for example, conversion into the [D1]-iminium ion where the incorporated
deuterium atom would lie in an equatorial position ([D1]-D2, Scheme 2). Previously obtained
crystal structures of proline-based enamines derived from ketones suggest that the more acidic
hydrogen atom is that whose abstraction leads directly to the enamine intermediate in which
the carboxylate group is anti to the C=C bond (Scheme 2, inset).18 In an investigation reported
by Hine and co-workers on the exchange reaction of [D6]-acetone in aqueous buffer catalyzed
by bifunctional primary–tertiary diamines, [D3]- and [D4]-acetone were formed at the very
beginning of the reaction, with [D5]-acetone not being detected.19 The pendant tertiary amine
base and the ability of the iminium ion derivative of acetone to undergo rapid rotation about
the C–C bonds contributed to this extremely efficient rate of exchange.
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most acidic and
sterically accessible site
Y
O
O
X
X
O
X
O
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H2
N
H1
H4
D
N
H3
Y
N
O
Y
H3
O
H2
H4
D2
[D1]-
The iminium ions are drawn in
analogy to the most stable
oxazolidinone, as detected in
organic solvents (see Ref 8e)
D
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2-
DPO4
imine
isomerization
-
slow
fast
Y
DHPO4
Y
X
X
-
D2PO4
D
O
H2
N
fast
H2
H4
N
H3
E
H3
D
O
O
H4
H3
H2
O
O
H4
2-
DPO4
D1
[D1]-
Scheme 2. Proposed selective abstraction of the axial hydrogen atom distal and anti to the
carboxylate substituent of the iminium intermediate.
The above data are consistent with the following mechanism (see Scheme 1): a)
Addition of the proline derivative to cyclohexanone to give the carbinoxyammonium A, which
−
2−
requires an acid (H2PO4 / HPO4 ) for conversion into carbinolammonium B; owing to steric
considerations, addition is likely to proceed through attack of the proline face opposite the
carboxylate group. b) Carbinolammonium B can then either be converted back into starting
material involving base-mediated deprotonation of the OH group, or, via its conjugate base,
carbinolamine C, undergo rate-determining acid-medicated dehydration to iminium D.
Similarly, Hine and co-workers have shown that acid-mediated dehydration of carbinolamines
derived from aldehydes and primary amines to give the corresponding iminium ion is rate
determining in acidic to neutral aqueous solution.20 c) In D2O, the corresponding OD group of
carbinolammonium B undergoes dedeuteronation at a slower rate owing to a primary isotope
effect, thus leading to greater partitioning of carbinolamine C into iminium D and a faster rate
of exchange, a situation that manifests as a large inverse solvent isotope effect. Large inverse
solvent isotope effects are rare but are observed in E1cB reactions, where rate-determining
expulsion of the β-positioned leaving group competes with protonation of the intermediate
carbanion.21 d) In the more stable chair conformer, the axial hydrogen atom distal and anti to
the proline carboxylate moiety of iminium D (Scheme 2) undergoes deprotonation to give
enamine E, which is ultimately transformed back into ketone via intermediates D, C, B. and A
at a rate faster than any other process that would lead to the exchange of any of the remaining
three hydrogen atoms.
The experimentally supported reaction coordinate suggests that the lower rate of
exchange of cyclohexanone in phosphate-buffered D2O in the presence of trans-4-
fluoroproline 3 might be due to differences in the transition state linking carbinolamine C and
iminium D. Specifically, dehydration of carbinolamine C derived from fluoroproline 3 may be
inhibited by a destabilizing Coulombic interaction between the electronegative fluorine atom,
which, owing to the gauche effect, will be moving towards an axial position, and the phosphate
species, which as it delivers a proton, will be becoming more anionic (Figure 3A). Presumably,
in imidazole buffer, the corresponding proton transfer from the cationic imidazolium ion does
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not present a differentiating impediment. A similar destabilizing interaction in the phosphate-
mediated protonation of carbinoxyammonium A (Scheme 3B) is likely to be less important
owing to the early transition state in this transformation. Note, it is reasonable to assume this
transformation (protonation of the carbinoxyammonium A) has an early transition state because
the reverse reaction (deprotonation of the carbonolammonium B) has been proposed to
contribute to the large inverse solvent isotope effect and thus would be deemed to proceed
through a late transition state.
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B
A
O
P
O
P
O
P
O
P
HO
O
HO
O
HO
O
HO
O
O
O
O
O
F
H
H
H
F
F
F
H
H
H2O
O
H
OH
O
N
O
N
H
N
N
H
H
O
O
O
O
O
O
O
B
D
TSA
TSC
B

D

Scheme 3. Destabilizing interactions in phosphate-mediated transformation of trans-4-
fluoroprolines intermediates in the hydrogen–deuterium exchange reaction of cyclohexanone
To further explore the effect of the identity and relative configuration of substituents at the 4-
position on the catalytic activity of proline derivatives, we prepared the cis and trans isomers
of both 4-dimethylammonium and 4-trimethylammonium proline (Figure 2) from
commercially available N-tert-butoxycarbonyl (N-Boc)-protected 4-hydroxyprolines.
D
D
N
N
N
N
CO2
CO2
CO2
CO2
N
N
N
N
D
D
D
D
D
D
D
D
5
4
(pKa 10.3, 7.4)
(pKa 10.3, 7.3)
6
7
(pKa 7.4)
(pKa 7.4)
Figure 2: Structures of aminoprolines 4–7.
The cis isomers, 4 and 6, were prepared from trans N-Boc-hydroxyproline methyl ester
(Scheme 4). Activation of the C4 hydroxy group as the mesylate followed by invertive
displacement with azide ion gave the cis 4-azidoproline derivative.22 Hydrogenative reduction
of the azide followed by reductive alkylation in the presence of formalin gave protected cis 4-
dimethylaminoproline 8, which was treated sequentially under basic and acidic conditions to
effect saponification and removal of the Boc group to give cis 4-dimethylaminoproline
dihydrochloride salt (4). Alternatively, N-methylation of dimethylaminoproline derivative 8
gave the trimethylammonium iodide derivative, which, owing to light sensitivity conferred by
the iodide ion on this compound and subsequent derivatives, was treated with ion-exchange
resin so that iodide could be replaced by chloride. Saponification and removal of the Boc group
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gave cis 4-trimethylammoniumproline salt 6. The trans isomers, 5 and 7, were prepared from
cis N-Boc-hydroxyproline by using an identical protocol (not depicted, see Experimental
Section).
7
8
9
HO
N3
1. MsCl
NEt3
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CO2Me
CO2Me
1. H2, Pd/C
N
Boc
N
Boc
2. NaN3
2. H2, Pd/C, CH2O
H
2 Cl
Cl
N
2 Cl
N
N
N
1. CH3I
1. NaOH
2. HCl
1. NaOH
2. HCl
CO2H
CO2Me
CO2Me
CO2H
N
H2
2. I  Cl
exchange
N
N
N
H2
Boc
Boc
6
, quant.
9
8
, 66%
, 88%
4
, quant.
Scheme 4: Synthesis of cis 4-aminoprolines 4 and 6.
pD profiles show that the trans diastereomers of dimethyl-ammonium proline 5 (pKa 7.3, 10.4)
and trimethylammonium proline 7 (pKa 7.3), are exceptionally active catalysts with maximum
rates ~10- and 5-fold higher than those for the reactions catalyzed by the corresponding cis
diastereomers, 4 and 6, which were of the level observed for fluoroproline 1 (Figure 3A,B).
Similar to the exchange reaction catalyzed by the fluoroprolines, reactions catalyzed by the
aminoprolines exhibited component-specific orders of ~1 for cyclohexanone, the proline
derivative, and phosphate/imidazole at pD values close to maximum rate. Further investigation
of reactions catalyzed by ammoniumproline 7 showed no substrate isotope effect and a large
solvent isotope effect (rate in D2O~9-fold greater than that in D2O/H2O, 1:1; Figure 3D).
Notably, although most pD–rate profiles exhibit a pronounced maximum that span a narrow
pD range, the pD–rate profile of 6 elicits a sharp rise in rate to a maximum value as the pD
approaches that corresponding to the pKa of 6 (7.4) and maintains this value in the pD range
7.9–10.5, before the rate falls gradually.
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Figure 3. (A) pD–rate profiles of reactions catalyzed by 4 and 5 in phosphate-buffered D2O
(I = 0.9). (B) pD–rate profiles of reactions catalyzed by 6 and 7 in phosphate-buffered D2O (I
= 0.9); (C) pD–rate profiles of reactions catalyzed by 6 and 7 in imidazole-buffered D2O (I =
0.15); (D) pD–rate profiles of reactions catalyzed by 7 in D2O/H2O (1:1; I = 0.9).
We believe that the origin of the exceptional levels of activity of trans 4-aminoprolines
5 and 7 is that nucleophilic attack of the proline derivative on the ketone would place the
resulting carbinoxy group in close proximity to the ammonium group at the 4-position of the
pyrrolidine ring (Scheme 5). The stabilizing Coulombic interaction between these two groups
should engender a relatively high concentration of carbinoxyammonium A and thus lead to a
relatively high rate of exchange. Electrostatic interactions, of the type proposed above, are
known to promote reactions involving the addition of nucleophiles to carbonyl compounds.
For example, the nonenzymatic hydrolysis of acetylcholine (2-trimethylammoniumethyl
acetate) in basic aqueous solution is significantly faster than that of ethyl acetate and there is
strong evidence that suggest that an electrostatic interaction between the oxyanion group and
the trimethylammonium group in the putative tetrahedral intermediate is the origin of this rate
enhancement.23 Aminoproline 5, which shows the highest level of activity, albeit only within
a narrow pD range close to its pKa value, would be expected to generate a higher concentration
of carbinoxyammonium A relative to that of 7 because of lower levels of steric hindrance.
There is no evidence to suggest that the presumed ability of the dimethylammonium group to
transfer a proton to the carbinoxy group contributes to a higher rate of the exchange reaction
catalyzed by 5; presumably, the reverse proton transfer (from oxygen to nitrogen) is so fast that
an external acid is still required. The rate-determining step for the reactions catalyzed by
prolines 5 and 7 appears to be the dehydration of carbinolamine C. However, the shape of the
pD profiles of the cis-dimethyl- and cis-trimethylammonium prolines, 4 and 6, is more
consistent with the rate-determining step being the acid-catalyzed addition of the proline
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derivative to cyclohexanone. The large inverse solvent isotope effect measured for the reactions
catalyzed by prolines 1 and 7 suggests that for the formation of the carbinolamine in these
reactions, the transfer of the proton from the acid to the alkoxide occurs quite late in the
transition, thus making this step insensitive to the pKa of the acid and only dependent on the
total amount of acid available. The observation that the rate associated with cis 4-
aminoprolines, 4 and 6, remains essentially constant over a wide pD range but drops
significantly at pD values higher than 10.5 (phosphate buffer) and 7 (imidazole buffer), at
which point the total concentration of acid will start to decrease, is consistent with this
hypothesis.
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The relatively sharp maxima of the pD profiles of catalysis by fluoroproline 1 (Figure
1A) and aminoprolines 5 and 7 (Figure 3A, B) deserves further comment. For example, the
exchange reaction catalyzed by fluoroproline 1 reaches a maximum rate at pD 7.4, close to the
pKa value of fluoroproline 1 (7.8), but falls rapidly as the pD value increases further. Although
the amount of fluoroproline available for covalent catalysis should continue to increase for a
region beyond pD 7.4 (at pD 7.8, 50% of the catalyst will still be in the ammonium form), the
overall strength of the available acids will tend to decrease and the overall strength of the
−
2−
available bases will tend to increase (as D2PO4 is being partitioned into DPO4 ; and 1.D into
the free amine 1—the catalyst also acts as donor and acceptor of deuterons). Therefore, the
overall rate constant associated with the base-catalysed conversion of carbinolammonium B
into A will increase and that associated with the acid-catalyzed conversion of carbinolamine C
into iminium ion D will decrease, thus causing the overall rate of exchange to fall. For
difluoroproline catalyst 1, the rates of exchange as a function of phosphate concentration have
been determined at pD 6.5 and pD 8.5 (I = 0.9). From the intercept of the concentration–
versus−rate plots, the minor contribution to the overall rate associated with the protonated form
of the catalyst acting as an acid (rather than phosphate buffer) can be ascertained. If these
−
−
contributions are subtracted, the relative contribution of [D2PO4 ] and [DPO4 ] can be
determined, with the assumption that the contribution of catalysis by [D3O+] is negligible.
Using the Henderson–Hasselbalch equation and the pKa values of aminocatalyst (7.8) and
−
D2PO4 (6.7), a value of ~33 is estimated for the relative contribution to the overall rate of
−
−
catalysis by the mono- and dianionic forms of the buffer, k(D2PO4 )/k(DPO4 ). Using these
values, the maximum of a simulated pD–rate profile for 1 (pD 7.4) agrees with the value
experimentally determined (see Supporting Information).
electrostatic
stabilization
N
Keq
N
O
D
O
N
N
D
O
O
O
O
A
Scheme 5. Proposed electrostatic interaction between the α-quaternary oxy-anion group and
the ammonium group on the 4-position of the pyrrolidine ring in carbinoxyammonium A
derived from either 5 or 7.
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CONCLUSIONS
9
Our findings provide fundamental insight on the design of efficient aminocatalytic systems for
transformations in water. Despite the highly stabilizing effect of water as solvent, inter- and
intramolecular electrostatic interactions involving charged and electron-rich atoms derived
from substrate, catalyst, and buffer can have a dramatic influence on catalytic activity. We
believe that cis- and trans-4-trimethyl ammoniumproline are especially promising in that
context.
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EXPERIMENTAL METHODS
General Methods. Procedures were performed at room temperature (~23 °C) unless
indicated otherwise. Reactions were monitored by thin-layer chromatography using
Whatman® aluminum-backed silica gel TLC plates with visualization by UV light or
ninhydrin staining. Compounds were purified by flash chromatography on silica gel, which
had a mesh of 230–400 (ASTM) and a pore size of 60 Å. The removal of solvents and other
volatile materials “under reduced pressure” refers to the use of a rotary evaporator at water-
aspirator pressure (<20 torr) and a water bath of <40 °C. Compounds “dried under vacuum”
refers to the placement of the material on a manifold connected to an oil-based pump capable
of maintaining pressures of <0.1 Torr. Fluoroprolines and protected forms of cis- and trans-
hydroxyprolines (for the preparation of aminoprolines) are commercially available.
Instrumentation. NMR spectra were acquired at ambient temperature with a Bruker
DMX-400 Avance spectrometer (1H, 400.1 MHz; 13C, 100.6 MHz) at the National Magnetic-
Resonance Facility at Madison (NMRFAM). The detection of 13C nuclei was proton
decoupled. 1H NMR spectra were referenced to TMS or to the residual solvent peak. 13C
NMR spectra were referenced to the residual solvent peak. Mass spectrometry was performed
with a Waters Micromass LCT®: electrospray ionization, time of flight; GCMS was
performed with a Shimadzu GCMS-QP2010S (gas chromatography, electron impact, single
quadrupole) in the Mass Spectrometry Facility in the Department of Chemistry at the
University of Wisconsin–Madison.
Synthesis of Aminoproline Derivatives. 1-tert-Butyl-2-methyl (2S,4S)-4-
(dimethylamino)pyrrolidine-1,2-dicarboxylate (8). A stream of Ar(g) was continuously passed
through a 250-mL 3-neck flask equipped with stirring bar. 10 % Pd/C (0.4 g) was added
followed by MeOH (50 mL). A solution of 1-tert-butyl-2-methyl-(2S,4S)-4-azidopyrrolidine-
1,2-dicarboxylate (3.15 g, 11.66 mmol)22 in MeOH (50 mL) was added, and the suspension
was cooled to 0 °C. The flask was evacuated and placed under a balloon of H2(g). The stirred
suspension was warmed to room temperature overnight. The balloon was removed, and the
suspension was placed under Ar(g). Formalin (2 mL) was added, and the flask was again
evacuated and placed under a balloon of H2(g). The resulting mixture was stirred overnight at
room temperature. The balloon was removed, and the suspension was carefully filtered through
Celite®; the residue was washed with MeOH (100 mL). The filtrate was evaporated, and the
resulting oil was taken up in CH2Cl2. The solution was washed with sat. aq. NaHCO3 (2  50
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mL), dried over Na2SO4(s), and evaporated. The residue was purified by silica gel column
chromatography (eluent: 1–2% MeOH/CH2Cl2). The resulting pale-yellow oil was placed
under house vacuum overnight (2.1 g, 7.72 mmol, 66% yield). H NMR (400 MHz, CDCl3,
1
7
8
9
mixture of rotamers 2:1; designated major (ma.) and minor (mi.) where possible):  = 1.40
(ma.) and 1.46 (mi.) (9H, s), 1.72–1.86 (1H, m), 2.17–2.24 (6H, m), 2.37–2.52 (1H, m), 2.54–
2.68 (1H, m), 3.13–3.24 (1H, m), 3.63–3.88 (4H, m), 4.19–4.32 (1H, m).13C NMR{1H} (100.6
MHz, CDCl3, mixture of rotamers; designated major (ma.) and minor (mi.) where possible): 
= 28.2 (ma.), 28.3 (mi.), 34.4 (mi.), 36.1 (ma.), 44.2 (mi.), 44.3 (ma.), 50.5 (ma.), 50.7 (mi.),
51.9 (ma.), 52.1 (mi.), 58.1 (mi.), 58.6 (ma.), 63.8 (ma.), 64.4 (mi.), 80.1 (ma. and mi.), 153.5
(ma.), 154.1 (mi.), 172.8 (mi.), 173.1 (ma.). HRMS (ESI-TOF)) m/z: [M + H]+ Calcd for
C13H25N2O4 273.1809; Found 273.1810.
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1-tert-Butyl-2-methyl-(2S,4S)-4-(trimethylammonium)pyrrolidine-1,2-dicarboxylate
Chloride (9). 1-tert-Butyl-2-methyl (2S,4S)-4-(dimethylamino)pyrrolidine-1,2-dicarboxylate
(8, 0.94 g, 3.45 mmol) was dissolved in CH2Cl2 (10 mL). MeI (1.07 mL, 17.26 mmol) was
added, and the resulting solution was allowed to stir at room temperature overnight. The
volatiles were then removed by rotary evaporation to give a foamy white solid. The solid was
dissolved in MeOH (5 mL) anionic ion exchange resin (Amberlite IRA410–chloride form) was
added (~15 equiv). The resulting suspension was allowed to stir for 10 min and was then
filtered; the resin was washed with a portion of MeOH (5 mL). The combined filtrates were
evaporated to give a white solid (0.98 g, 3.04 mmol, 88% yield). 1H NMR (400 MHz, CDCl3,
mixture of rotamers, broad signals):  = 1.40-1.46 (9H, m), 2.26–2.37 (1H, m), 2.86–2.96 (1H,
m), 3.44–3.50 (9H, m), 3.57–3.65 (1H, m), 3.71–3.75 (3H, m), 3.98–4.06 (1H, m), 4.32–4.40
(1H, m), 4.76–4.90 (1H, m).13C NMR{1H} (100.6 MHz, CDCl3, broad signals):  = 28.1, 30.3,
45.3, 52.8, 53.5, 57.1, 70.0, 81.5, 152.9, 171.6. HRMS (ESI-TOF)) m/z: [M ]+ Calcd for
C14H27N2O4 287.1966; Found 287.1974.
1-tert-Butyl-2-methyl-(2S,4R)-4-(dimethylamino)pyrrolidine-1,2-dicarboxylate (10). A
stream of Ar(g) was continuously passed through a 250-mL 3-neck flask equipped with stirring
bar. 10% Pd/C (0.4 g) was added followed by MeOH (50 mL). A solution of 1-tert-butyl-2-
methyl-(2S,4R)-4-azidopyrrolidine-1,2-dicarboxylate (3.22 g, 11.92 mmol)22,24 in MeOH
(50 mL) was added, and the suspension was cooled to 0 ºC. The flask was evacuated and placed
under a balloon of H2(g). The stirred suspension was warmed to room temperature overnight.
The balloon was removed, and the suspension was placed under Ar(g). Formalin (2 mL) was
added, and the flask was again evacuated and placed under a balloon of H2(g). The resulting
mixture was stirred overnight at room temperature. The balloon was removed, and the
suspension was carefully filtered through Celite®; the residue was washed with MeOH
(100 mL). The filtrate was evaporated, and the resulting oil was taken up in CH2Cl2. The
solution was washed with sat. aq. NaHCO3 (2  50 mL), dried over Na2SO4(s), and evaporated.
The residue was purified by silica gel column chromatography (eluent: 1–2% MeOH/CH2Cl2).
The resulting pale-yellow waxy solid was placed under house vacuum overnight (2.45 g,
1
9.00 mmol, 76% yield). H NMR (400 MHz, CDCl3, mixture of rotamers 60:40; designated
major (ma.) and minor (mi.) where possible):  = 1.38 (ma.) and 1.44 (mi.) (9H, s), 2.00–2.23
(8H, m), 2.73–2.88 (1H, m), 3.08–3.22 (1H, m), 3.65–3.83 (4H, m), 4.30 (ma.) and 4.39 (mi.)
(1H, m). 13C NMR{1H} (100.6 MHz, CDCl3, mixture of rotamers; designated major (ma.) and
minor (mi.) where possible):  = 28.2 (ma.), 28.3 (mi.), 34.0 (mi.), 35.0 (ma.), 44.1 (ma. and
mi.), 50.3 (ma.), 50.6 (mi.), 52.0 (ma.), 52.1 (mi.), 58.2 (mi.), 58.7 (ma.), 62.8 (ma.), 63.6 (mi.),
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80.0 (ma. and mi.), 153.5 (ma.), 154.2 (mi.), 173.1 (mi.), 173.4 (ma.). HRMS (ESI-TOF)) m/z:
[M + H]+ Calcd for C13H25N2O4 273.1809; Found 273.1805.
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7
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9
1-tert-Butyl-2-methyl-(2S,4R)-4-(trimethylammonium)pyrrolidine-1,2-dicarboxylate
chloride (11). 1-tert-Butyl-2-methyl (2S,4R)-4-(dimethylamino)pyrrolidine-1,2-dicarboxylate
(10, 0.93 g, 3.41 mmol) was dissolved in CH2Cl2 (10 mL). MeI (1.07 mL, 17.26 mmol) was
added, and the resulting solution was allowed to stir at room temperature overnight. The
volatiles were then removed by rotary evaporation to give a foamy white solid. The solid was
dissolved in MeOH (5 mL), and anionic ion exchange resin (Amberlite IRA410–chloride form)
was added (~15 equiv). The resulting suspension was allowed to stir for 10 min and was then
filtered; the resin was washed with a portion of MeOH (5 mL). The combined filtrates were
evaporated to give a white solid (1.03 g, 3.2 mmol, 94% yield). 1H NMR (400 MHz, CDCl3,
mixture of rotamers 2:1; designated major (ma.) and minor (mi.) where possible):  = 1.35
(ma.) and 1.43 (mi.) (9H, m), 2.47–2.58 (1H, m), 2.82–3.02 (1H, m), 3.45–3.53 (9H, m), 3.64–
3.78 (4H, m), 3.87–3.96 (1H, m), 4.46–4.56 (1H, m), 4.57–4.76 (1H, m).13C NMR{1H} (100.6
MHz, CDCl3, mixture of rotamers designated major (ma.) and minor (mi.) where possible): 
= 28.1 (ma.), 28.3 (mi.), 29.8 (mi.), 30.7 (ma.), 45.4 (ma. and mi.), 52.7 (ma. and mi.), 53.0
(ma.), 53.5 (mi.), 57.6 (mi.), 57.8 (ma.), 71.2 (ma.), 72.0 (mi.), 81.4 (ma.), 81.7 (mi.), 153.0
(ma.), 153.4 (mi.), 171.7 (mi.), 171.8 (ma.). HRMS (ESI-TOF)) m/z: [M ]+ Calcd for
C14H27N2O4 287.1966; Found 287.1974.
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(2S,4S)-4-(dimethylamino)pyrrolidine-2-carboxylic Acid Dihydrochloride (4·2HCl). 1-
tert-Butyl-2-methyl-(2S,4S)-4-(dimethylamino)pyrrolidine-1,2-dicarboxylate (8, 1.17 g,
4.30 mmol) was dissolved in 1,4-dioxane (10 mL). 1 M NaOH in H2O (4.5 mL, 4.5 mmol) was
added, and the resulting solution was allowed to stir at room temperature overnight. The
volatiles were then removed by rotary evaporation. The solid was redissolved in 4 M HCl in
1,4-dioxane (10 equiv, 10.8 mL), and the resulting solution was allowed to stir for 30 min at
room temperature. The volatiles were removed by rotary evaporation to leave a white solid (1.0
g, 4.3 mmol, quantitative). NMR analysis showed that the compound was pure. 1H NMR (400
MHz, D2O):  = 2.28 (1H, ddd, J = 12.5, 10.7, 9.5 Hz), 2.84 (3H, s), 2.85 (3H, s), 2.92 (1H,
ddd, J = 12.5, 7.6, 7.6 Hz), 3.55 (1H, dd, J = 12.9, 8.9 Hz), 3.86 (1H, dd, J = 12.9, 8.5 Hz),
4.14 (1H, dddd, J = 9.5, 8.9, 8.5, 7.6 Hz), 4.50 (1H, dd, J = 10.7, 7.6 Hz). 13C NMR{1H} (100.6
MHz, D2O):  = 29.5, 42.2, 42.6, 45.0, 58.3, 62.9, 169.2. HRMS (ESI-TOF)) m/z: [M+1]+
Calcd for C7H15N2O2 159.1123; Found 159.1129.
(2S,4R)-4-(Dimethylamino)pyrrolidine-2-carboxylic acid dihydrochloride (5·2HCl).
1-tert-Butyl-2-methyl-(2S,4R)-4-(dimethylamino)pyrrolidine-1,2-dicarboxylate (10,
0.92 g, 3.39 mmol) was dissolved in 1,4-dioxane (10 mL). 1 M NaOH in H2O (3.56 mL, 3.56
mmol) was added, and the resulting solution was allowed to stir at room temperature overnight.
The volatiles were then removed by rotary evaporation. The solid was redissolved in 4 M HCl
in 1,4-dioxane (10 equiv, 8.5 mL), and the resulting solution was allowed to stir for 30 min at
room temperature. The volatiles were removed by rotary evaporation to leave a white solid
(0.79 g, 3.39 mmol, quantitative). NMR analysis showed that the compound was pure. 1H NMR
(400 MHz, D2O):  = 2.61 (1H, ddd, J = ~15, 8.8, 8.8 Hz), 2.78 (1H, ddd, J = ~15, 7.5, 7 Hz),
2.90 (6H, br. s), 3.60 (1H, dd, J = 12.6, 8.5 Hz), 3.98 (1H, dd, J = 12.6, 8.2 Hz), 4.16 (1H,
13
dddd, J = 8.8, 8.5, 8.2, 7.5 Hz), ~4.65 (1H, multiplet obscured by residual solvent). C
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NMR{1H} (100.6 MHz, D2O):  = 29.6, 42.2, 42.4, 45.4, 58.8, 62.6, 170.0. HRMS (ESI-TOF))
m/z: [M+Na]+ Calcd for C7H14N2NaO2 181.0953; Found 181.0947.
6
7
(2S,4S)-4-(Trimethylammonium)pyrrolidine-2-carboxylic
Acid
Chloride
Hydrochloride (6·2HCl). 1-tert-Butyl-2-methyl-(2S,4S)-4-(trimethylammonium)pyrrolidine-
1,2-dicarboxylate chloride (9, 0.98 g, 3.04 mmol) was dissolved in 1,4-dioxane (10 mL). 1 M
NaOH in H2O (3.2 mL, 3.19 mmol) was added, and the resulting solution was allowed to stir
at room temperature overnight. The volatiles were then removed by rotary evaporation. The
solid was redissolved in 4 M HCl in 1,4-dioxane (10 equiv, 7.6 mL), and the resulting solution
was allowed to stir for 30 min at room temperature. The volatiles were removed by rotary
evaporation to leave a white solid (0.75 g, 3.04 mmol, quantitative). NMR analysis showed
that the compound was pure. 1H NMR (400 MHz, D2O, pD = 5.1, 150 mM phosphate, I = 0.9):
 = 2.37 (1H, m), 2.87 (1H, m), 3.17 (9H, s), 3.74 (1H, m), 3.88 (1H, m), 4.27 (1H, m), 4.48
(1H, m). 13C NMR{1H} (100.6 MHz, D2O, pD = 5.1, 150 mM phosphate, I = 1.5):  = 28.5,
43.0, 51.9, 59.9, 70.8, 171.6. HRMS (ESI-TOF)) m/z: [M]+ Calcd for C8H17N2O2 173.1285;
Found 173.1283.
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(2S,4R)-4-(trimethylammonium)pyrrolidine-2-carboxylic
Acid
Chloride
Hydrochloride (7·2HCl). 1-tert-Butyl-2-methyl-(2S,4R)-4-(trimethylammonium)pyrrolidine-
1,2-dicarboxylate chloride (11, 1.03 g, 3.20 mmol) was dissolved in 1,4-dioxane (10 mL). 1 M
NaOH in H2O (3.4 mL, 3.40 mmol) was added, and the resulting solution was allowed to stir
at room temperature overnight. The volatiles were then removed by rotary evaporation. The
solid was redissolved in 4 M HCl in 1,4-dioxane (10 equiv, 8.0 mL), and the resulting solution
was allowed to stir for 30 min at room temperature. The volatiles were removed by rotary
evaporation to leave a white solid (0.79 g, 3.20 mmol, quantitative). NMR analysis showed
that the compound was pure. 1H NMR (400 MHz, D2O):  = 2.72–2.80 (2H, m), 3.13 (9H, s),
3.75 (1H, dd, J = 13.2, 8.4 Hz), 3.97 (1H, dd, J = 13.2, 8.7 Hz), 4.45 (1H, dddd, J = 8.7, 8.4,
~8, ~8 Hz), ~4.70 (1H, multiplet obscured by residual solvent). 13C NMR{1H} (100.6 MHz,
D2O):  = 27.8, 43.7, 52.0, 58.5, 69.9, 169.4. HRMS (ESI-TOF)) m/z: [M]+ Calcd for
C8H17N2O2 173.1285; Found 173.1281.
Determination of pKa Values. The logarithmic acidity constants (pKa values) of the
proline derivatives used in this study in environments that are relevant and normal to the
1
reaction under investigation (D2O, 150 mM phosphate, I = 0.9) were determined using H
NMR spectroscopy. Acidic and alkaline 30 mM D2O solutions of the proline derivative under
investigation were prepared by adding the required amount of catalyst (as its HCl salt) to
150 mM Na3PO4 (I = 0.9) and 150 mM D3PO4 (ionic strength brought to I = 0.9 using NaCl)
respectively. A pH probe was placed in the alkaline solution (5 mL) with stirring bar, and the
pH meter output was read and noted. The pD value was obtained by adding 0.41 to the pH-
meter value.25 Approximately 0.7 mL was removed from the solution and placed in an NMR
1
tube. A H NMR spectrum was recorded. The NMR solution was returned to the pH-probed
stirred vessel. The acidic solution was added dropwise to the pH-probed stirred solution until
the pH-meter value (pD value) dropped by an amount not exceeding 0.25–0.50 units. The pH-
meter value/pD value was again noted. Again, approximately 0.7 mL of the solution was
transferred to an NMR tube and an NMR spectrum was recorded. The NMR solution was
returned to the pH-probed stirred vessel. The procedure was repeated until such time as the pH
meter value/pD value falls to well below (3 to 4 units) the expected pKa value of the proline
derivative under investigation. The NMR spectra obtained at the various pH meter/pD values
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was compiled and where possible the chemical shifts of all protons (in Hz) in all spectra were
noted in a spreadsheet in which each row represented a spectrum taken at a particular pD value,
and the columns represented chemical shifts of the specific protons. Using these data, the
difference in chemical shifts (Δδ in Hz) of an appropriate pair of protons was calculated and
plotted against the associated pD value. An appropriate pair of protons was deemed to be that
where the associated chemical shifts could be determined accurately (that is, not obscured in
any way within the pD range measured) and where the Δδ over the pD range was as large as
possible (thus reducing the error). Using GraphPad Prism software, the plot was fitted with the
best sigmoidal curve. The pD value at the inflection point of the fitted curve represents the pKa
value of the proline derivative. The value was verified by plotting the Δδ of an alternative pair
of protons against pD.
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Determination of the Rate of Deuterium/Hydrogen Incorporation as a Function of
pD/pH. Preparation of Buffers. 150 mM phosphate D2O buffers of constant ionic strength
(I = 0.9) of various pD values were prepared as follows. A pH-meter probe was immersed in a
10-mL Falcon® tube containing 5 mL of a 150 mM D2O solution of Na3PO4 (I = 0.9). A
150 mM D2O solution of D3PO4 (I = 0.9, NaCl) was added dropwise until the desired pD value
was reached (pD value = pH-meter value + 0.41). For the preparation of 150 mM imidazole
D2O buffers of constant ionic strength (I = 0.150) of various pD values, the above procedure
was followed using 150 mM D2O solutions of imidazole (with 0.150 mM of NaCl) and
imidazole DCl salt. For the preparation of 150 mM phosphate D2O/H2O (1:1) buffers of
constant ionic strength (I = 0.9) at various pH-meter reading values, the above procedure was
followed using a 150 mM H2O/D2O (1:1) solution of Na3PO4 and a 150 mM H2O/D2O (1:1)
solution of D3PO4 (I = 0.9, NaCl).
1H NMR Method for Determination of the Rate of Deuterium Incorporation. A 150 mM
D2O solution of phosphate of the pD value under investigation (1 mL) was placed in a 5-mL
vial. Cyclohexanone (20 μL, 0.193 mmol) was added, and the capped vial was vortexed for 10
s. A D2O solution of the catalyst under investigation (an amount representing approximately
10 mol% (0.0193 mmol) with an added volume of no greater than 50 µL] was added, and the
resulting mixture was vortexed for 10 s. A 0.7-mL aliquot of the homogeneous solution was
immediately placed in an NMR tube, and the tube was inserted into the NMR probe. 1H NMR
spectra were recorded at constant intervals. Using Bruker software, the multizg function was
employed with the number of scans (ns) was set to 4, and the number of dummy scans (ds) set
to a value between 20 and 70 (depending upon the rate of reaction); between 10 and 50
consecutive acquisitions were recorded wherein the ds value represents the interval between
acquisitions. The exact time between acquisitions was determined by examining the time
interval between the storage of consecutive FID files. The resultant FID files were processed
using MestReNova software. The number of moles of deuterium nuclei per liter of reaction
solution incorporated into the α positions of cyclohexanone was calculated by subtracting the
integration of the α protons from that of the β protons (the integration of the β protons
corresponds to 0.736 M; 0.193 mmol of cyclohexanone in ~1.050 mL of solution represents
0.184 M, multiplied by 4 because there are 4 α and 4 β protons). In some samples, signals from
the catalyst overlapped with the α protons of cyclohexanone; in those cases, the appropriate
correction was made. The amount of deuterium incorporated (M) was plotted against the period
of time between the start of the reaction, and the time of acquisition (s); the first acquisition
was set at t = 0 s. Spectra where 10% of possible deuterium incorporation was exceeded were
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discarded. The initial rates (M·s–1) were determined by extracting the slope from the best line
to fit the data (using GraphPad Prism software). For each catalyst the initial rate calculated at
each pD value was plotted against pD.
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Determination of Rate Laws. For each catalyst, the rate-law exponent of each of the
three reaction components (namely catalyst, ketone and buffer) was determined by measuring
the rate of deuterium incorporation at various concentrations of the component under
investigation whilst maintaining constant the concentration of the remaining components. The
rates were measured using 1H NMR spectroscopy according to the procedure outlined above.
The log of the rate was then plotted against the log of the concentration of the component being
varied; the slope of the resulting line represents the pertinent exponent. Because the mechanism
and/or the identity of the turnover-rate-determining transition state/intermediate could depend
on the pD value of the solution, the exponents were usually determined at a pD value where
catalysis was most efficient for the particular catalyst under investigation. The exponent of the
catalyst and the ketone were determined at 0.15 M phosphate (I = 0.9). To obtain the exponent
of the buffer, D2O solutions of varying concentration of phosphate (0.05–0.5 M) of the same
ionic strength (I = 3.0) and pD value were prepared. These solutions were prepared as follows:
D2O solutions of 0.5 M Na3PO4 (I = 3.0) and 0.5 M D3PO4 (I = 3.0, NaCl) were prepared.
Through appropriate dilution of aliquots of these solutions with D2O (3.0 M NaCl), solutions
of Na3PO4 and D3PO4 of varying concentration of phosphate (0.05 to 0.5 M in intervals of 0.05
M) and constant ionic strength (I = 3.0) were prepared. Using a pH meter, the appropriate pairs
of acidic and basic solutions were combined to obtain a solution of a particular concentration
of phosphate at a particular pD value. The background rate (catalysis of exchange by
aminocatalyst alone) was subtracted from the observed rate to obtain an exponent that was
predominantly associated with iminium/enamine/phosphate catalysis by the proline derivative.
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Investigation of Substrate Isotope Effects. The rate of incorporation of hydrogen and
deuterium into [D0]cyclohexanone and [D4]cyclohexanone, respectively, as catalyzed by the
proline derivative under investigation, were determined in 1:1 mixtures of phosphate-buffered
(150 mM, I = 0.9) D2O and H2O of a particular pH-meter reading. The use of a common
reaction medium for the comparison of rates of the exchange reaction of [D0]cyclohexanone
and that of [D4]cyclohexanone reduces errors and, more importantly, allows the isotope effects
pertaining to species derived from the solvent, the buffer, and the substrate to be disentangled.
The D2O and H2O buffers were prepared according to the method described above and then
mixed in the required ratio (1:1; total volume: 1 mL). A D2O/H2O solution (1:1; total volume:
70 μL) of the catalyst (0.0193 mmol) was added to the mixture. [D0]Cyclohexanone (0.193
mmol, 20 µL) was then added, and the mixture was shaken vigorously for 10 s to ensure
homogeneity; at the same time, a stopwatch was started. For a period ranging from 1–2 h, at
intervals ranging from 10–20 min, 100 µL of the mixture was removed and added to a vial
containing 1.0 mL of CH2Cl2 and suspended sodium sulfate. The vial was sealed and shaken.
The resulting solution of CH2Cl2 was decanted and passed through a short silica plug, which
was prepared using a Pasteur pipette, cotton wool, silica gel, sand, and when prepared
contained approximately 2–3 cm of silica gel. The filtrate was placed in a labelled vial suitable
for GS/MS analysis. When the experiment was complete, the prepared vials, together with a
vial containing a solution of fresh [D0]cyclohexanone in CH2Cl2 as a standard, were analyzed
using GS/MS. The above procedure was repeated using [D4]cyclohexanone. For each vial, the
peak corresponding to the mixture of cyclohexanone isotopes was integrated, and the relative
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amounts of each mass ion tabulated in an Excel spreadsheet. For the experiments using
[D0]cyclohexanone (M=98), integrations corresponding to a species (M=97) and its associated
M+n ions (n = 1 and 2), which were calculated assuming natural abundance, were subtracted.
The resultant integration values associated with M=98, M=99, and M=100 were assumed to
represent the sum of the integrations associated with [D0]cyclohexanone, [D1]cyclohexanone,
and [D2]cyclohexanone of both natural and non-natural (derived from the reaction) abundance.
The relative amounts of [D1]cyclohexanone and [D2]cyclohexanone, as derived from the
reaction, expressed as a percentage of the sum of the amounts corresponding to [D0]-, [D1]-,
and [D2]cyclohexanone, were determined by sequential subtraction. The relative amounts of
[D1]cyclohexanone and [D2]cyclohexanone of non-natural abundance were added and
converted into moles of deuterium incorporated, and the values were plotted against time (in
seconds). A similar analysis was done for the incorporation of hydrogen into
[D4]cyclohexanone. The slopes of the resulting graphs were the expressed as a ratio to
determine kH/kD.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at
DOI: 10.1021/acs.joc.xxxxxxx. NMR spectral data of catalysts, pKa data, kinetic data and plots,
(PDF).
AUTHOR INFORMATION
Corresponding Authors
*E-mail: R.T.R., rtraines@mit.edu; E.L.M., eddie.myers@nuigalway.ie
ORCID
Ronald T. Raines: 0000-0001-7164-1719
Eddie L. Myers: 0000-0001-7742-4934
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
E.M. acknowledges support from the School of Chemistry, NUI–Galway. M.J.P. was
supported by Molecular and Cellular Pharmacology Training Grant T32 GM008688 (NIH) and
predoctoral fellowship 09PRE2260125 (American Heart Association). This work was
supported by grants R01 AR044276 and R01 GM044783 (NIH). NMRFAM was supported by
grant P41 GM103399 (NIH).
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