Dissecting the mechanisms of a class of chemical glycosylation using primary 13C kinetic isotope effects

Article abstract of DOI:10.1038/nchem.1404

Although arguably the most important reaction in glycoscience, chemical glycosylations are among the least well understood of organic chemical reactions, resulting in an unnecessarily high degree of empiricism and a brake on rational development in this critical area. To address this problem, primary ¹³C kinetic isotope effects have now been determined for the formation of β- and α-manno- and glucopyranosides using a natural abundance NMR method. In contrast to the common current assumption, for three of the four cases studied the experimental and computed values are indicative of associative displacement of the intermediate covalent glycosyl trifluoromethanesulfonates. For the formation of the α-mannopyranosides, the experimentally determined KIE differs significantly from that computed for an associative displacement, which is strongly suggestive of a dissociative mechanism that approaches the intermediacy of a glycosyl oxocarbenium ion. The application of analogous experiments to other glycosylation systems should shed further light on their mechanisms and thus assist in the design of better reactions conditions with improved stereoselectivity.

Full text of DOI:10.1038/nchem.1404

ARTICLES
PUBLISHED ONLINE: 22 JULY 2012 | DOI: 10.1038/NCHEM.1404
Dissecting the mechanisms of a class of
chemical glycosylation using primary
13C kinetic isotope effects
1
2,3
1
1
2,3
and David Crich1,4
´
Min Huang , Graham E. Garrett , Nicolas Birlirakis , Luis Bohe , Derek A. Pratt
*
*
Although arguably the most important reaction in glycoscience, chemical glycosylations are among the least well
understood of organic chemical reactions, resulting in an unnecessarily high degree of empiricism and a brake on rational
development in this critical area. To address this problem, primary 13C kinetic isotope effects have now been determined
for the formation of b- and a-manno- and glucopyranosides using a natural abundance NMR method. In contrast to the
common current assumption, for three of the four cases studied the experimental and computed values are indicative of
associative displacement of the intermediate covalent glycosyl trifluoromethanesulfonates. For the formation of the
a-mannopyranosides, the experimentally determined KIE differs significantly from that computed for an associative
displacement, which is strongly suggestive of a dissociative mechanism that approaches the intermediacy of a glycosyl
oxocarbenium ion. The application of analogous experiments to other glycosylation systems should shed further light
on their mechanisms and thus assist in the design of better reactions conditions with improved stereoselectivity.
he perceived importance of oligosaccharides in biology and of an important glycosylation reaction and its dependence on
the potential for their exploitation in medicine has made substitution pattern.
T
glycoscience one of the most vibrant and dynamic fields at
the interface of chemistry and biology1–4. Because of the difficulties Results and discussion
in obtaining pure, homogeneous samples of oligosaccharides from Our demonstration that the 4,6-O-benzylidene-directed b-manno-
natural sources, glycochemistry is a critical and fundamental com- pyranosylation reaction20–22 proceeds via the coupling of a highly
ponent of glycoscience5–8. Glycochemistry turns on the critical reactive a-mannosyl triflate23,24 with an acceptor alcohol without
hub of glycosidic bond formation9,10, yet, paradoxically, the art of the need for a promoter provided a rare opportunity to study an
glycosidic bond formation is one of the least well understood11,12 important class of glycosylation reaction in detail. The rapidity of
and most empirical13 in the science of organic chemistry.
thereactionatlowtemperaturesandtheabsenceofasuitablechromo-
Excepting so-called alternative methods10, essentially all discussions phore excluded the possibilityof simple kinetic measurements for the
of glycosidic bond formation invoke the glycosyl oxocarbenium determination of reaction order and focused our attention on the
ion14,15, but this oft-computed16–18 key intermediate has yet to measurement of KIEs by NMR methods25–27, preferably at natural
be observed directly14,19. In reality, glycosylation reactions are abundancesoastoavoidthetime-consumingandexpensivesynthesis
probably best considered as taking place via a range of mechanisms of labelled compounds28,29. Problems of sensitivity and resolution
1
with more or less tightly associated transition states between the (500 MHz H operating frequency) limited our earlier work to the
two formal extremes of the SN1 and SN2 pathways, with the observation of secondary 2H KIEs and even then required the labor-
position of any particular example on this spectrum determined ious synthesis of samples enriched in deuterium30. With an 800 MHz
by a number of factors including protecting groups, solvents, (1H operating frequency) NMR spectrometer and cryogenic probe
leaving group and promoter14,15. In the absence of any direct technology at our disposal, we have now succeeded in determin-
observation of glycosyl oxocarbenium ions we fall back on indirect ing primary 13C KIEs at natural abundance for benzylidene-directed
methods and reaction kinetics, but these are typically complicated glycosylation reactions in the glucose and mannose series and show
by the (at least) three-component nature of most glycosylation that mechanism is a function of donor stereochemistry and in the
systems that involve a glycosyl donor, an acceptor and a promoter. mannose series differs for the two anomeric products.
We report here on the use of primary 13C kinetic isotope
The 4,6-O-benzylidene-protected mannosyl sulfoxide 1 was pre-
effect (KIE) measurements, in which the extent of 12C/13C isotopic pared as previously described23 and converted at 272 8C in dichloro-
fractionation at the anomeric position in the course of a methane solution to the a-glycosyl triflate 2 by the addition of an
glycosylation reaction is determined and, with the aid of excess of trifluoromethanesulfonic anhydride (Tf2O) in the presence
quantum chemical calculations, used to assign more or less tightly of 2,4,6-tri-tert-butyl pyrimidine (TTBP)31 as a mild, hindered,
associated transition states to particular cases. To avoid the need non-nucleophilic base. The acceptor, 2-propanol, was then added
for synthesis of isotopically enriched substrates, the experiment in quantities to ensure partial conversion of triflate 2 to a- and
was conducted at natural abundance by very high field NMR b-glycosides 3 and 4 before the reaction was quenched at –72 8C
spectroscopy (200 MHz operating frequency for 13C measurement). by the addition of saturated aqueous NaHCO3 (Table 1). After
Overall, the technique provides insight into the mechanism work up, the extent of conversion was determined by integration of
1Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France, 2Department of
Chemistry, Queen’s University, Kingston, Ontario, Canada K7L 4V1, 3Department of Chemistry, University of Ottawa, Ottawa, Ontario, Canada K1N 6N5,
4Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA. e-mail: dpratt@uottawa.ca; dcrich@chem.wayne.edu
*
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© 2012 Macmillan Publishers Limited. All rights reserved.
663

NATURE CHEMISTRY DOI: 10.1038/NCHEM.1404
ARTICLES
competing triflation of the alcohol, which reduces its concentration
and so retards the bimolecular reaction. This is consistent with
formation of the a-product being zero order in alcohol (SN1), with
that of the b-product depending on alcohol concentration (SN2)
and overall with the KIE measurements.
Table 1 | Experimentally determined primary 13C KIEs for
a- and b-mannopyranosides 3 (entries 1–5) and 4
(entries 6–10).
OMe
O
OMe
O
Ph
O
Tf2O (promoter)
TTBP
Ph
O
O
O
In contrast to mannopyranosylation21,22, in the glucopyranose
series the presence of a 4,6-O-benzylidene acetal results in preferen-
tial a-glycoside formation, most probably reflecting a shift in mech-
anism. We therefore examined the formation of a- and b-glucosides
7 and 8 from sulfoxides 5, via the triflate 6, by the same 13C NMR
KIE technique, leading to the data presented in Table 232. Unlike the
mannopyranosides, the observed 13C KIEs in the glucose series are
at the lower end of the range expected for a bimolecular reaction for
both the a- and b-series. As the formation of mannosyl and glucosyl
triflates from sulfoxides is unaffected by either the anomeric
configuration or the configuration at sulfur of the sulfoxides
employed21–24, for preparative reasons the equatorial sulfoxides 5
were used for the work in the glucose series (Table 2), whereas
the axial sulfoxide 3 was used in the mannose series (Table 1).
To complement the experimental data, and assist in its interpret-
ation, we carried out density functional theory computations of
possible transition-state structures for glycosylation. We selected
the B3LYP/6-311þþG(3df,3pd)//B3LYP/6-31G(d,p) level32–34 as
a compromise between speed and accuracy as it is sufficient for
reproduction of the anomeric effect16, but fast enough to survey
the dozens of possible trajectories for the reactions of both
anomers of each glycosyl donor. We also used a self-consistent reac-
tion field throughout the structure optimizations to capture any
bulk medium effects imposed by the solvent used in the experiments
above. The four lowest-energy transition-state structures identified
for the formation of a- and b-mannosides and the corresponding
glucosides are shown in Fig. 1a–d, together with the calculated
13C (and 2H) KIEs (which were indistinguishable whether calculated
using the Bigeleisen–Mayer equation or the ratio of rate constants
calculated using transition state theory)35, pertinent atomic
MeO
MeO
Ph
CH2Cl2, –72 °C
S
+
OTf
_
O
1 (Mannosyl donor)
2 (Mannosyl triflate)
OMe
O
OMe
O
Ph
Ph
iPrOH (acceptor)
O
O
O
O
+
OiPr
MeO
MeO
OiPr
3 (α-Mannoside)
4 (β-Mannoside)
Entry
Tf2O (equiv.)
Conversion (%)
a:b
KIE (25 8C)*†
a-Mannopyranoside 3
1
2
3
4
1.5
11.4
16.0
8.9
13.5
20.9
1:4
1.006
1.004
1.006
1.006
2.4
2.4
2.5
2.5
1:3.5‡
1:2.7
1:2.2
1:1.7
5
1.002
Average
1.005+0.002
b-Mannopyranoside 4
6
7
8
9
1.5
45.1
55.6
24.1
30.3
34.7
1:4
1.026
1.019
1.022
1.021
2.4
2.4
2.5
2.5
1:3.5‡
1:2.7
1:2.2
1:1.7
10
1.025
Average
1.023+0.003
*All KIEs were measured at –72 8C and were converted to 25 8C assuming
KIE258C ¼ exp{(201/298)ln(KIE272 8C)} (refs 27,29). †Errors quoted in the average values are at 1s
(see Supplementary Tables S1–S4 for details). ‡The higher a:b ratio for this entry with respect to
entries 3 and 4, despite the comparable amount of Tf2O used, presumably reflects the reaction of
adventitious water with Tf2O, which lowers its effective concentration. See text for a discussion of
the effect of Tf2O concentration.
1
the H NMR spectrum of the crude reaction mixture (800 MHz)
Table 2 | Experimentally determined primary 13C KIEs for a-
and b-glucopyranosides 7 (entries 1–4) and 8 (entries 5–8).
against the internal standard 4,4,5,5-tetramethyl-2-(1-naphthyl)-
1,3-dioxolane, and the products isolated chromatographically.
The 13C NMR spectra of the two anomeric products were then
recorded and the integrals were determined for the remote
benzylidene carbon, used as an internal standard, and the anomeric
carbon, in both the products, and compared with those previously
determined in the same manner for the starting sulfoxide. Use of
the standard equation for KIE determination based on the
observation of reaction products (equation (1)), where F1 is the
fractional conversion and R0 and Rp are the ratio of the anomeric
and benzylidene carbon integrals in the substrate and product,
respectively27, afforded the results presented in Table 1 for the
a- and b-mannopyranoside products.
_
O
Ph
O
Ph
O
Tf2O (promoter)
TTBP
O
O
O
O
+
Ph
S
MeO
MeO
MeO
MeO
CH2Cl2, –72 °C
OTf
5 (Glucosyl donor)
6 (Glucosyl triflate)
Ph
O
O
Ph
O
iPrOH (acceptor)
O
O
O
+
OiPr
MeO
MeO
MeO
MeO
OiPr
7 (α-Glucoside)
8 (β-Glucoside)
a:b
KIE (25 8C)*†
ln(1 − F1)
k
k
12C
13C
Entry
Tf2O (equiv.)
Conversion (%)
KIE =
=
(1)
a-Glucopyranoside 7
ln[1 − (F1Rp/R0)]
1
2
3
4
1.2
1.2
2.5
1.2
31.4
9.8
7.1
1:0.5
1.029
1.025
1.015
1.022
1:0.7
1:1.7
1:1
Comparison of the results presented in Table 1 reveals that the
primary 13C KIE values for the a- and b-mannoside products are dis-
tinctly different, suggesting different mechanisms for the formation
of the two anomers. The values for b-anomer 4 lie close to the
range (1.03–1.08) expected for a bimolecular (SN2) reaction25,
whereas those for a-anomer 3 are closer to the expected values
(1.00–1.01) for a unimolecular (SN1) reaction25. It is also evident
from inspection of Table 1 that the ratio of the two products
depends on the amount of Tf2O employed, with lower b:a ratios
occurring when more Tf2O is used. We interpret this observation,
which was deliberately exploited to obtain sufficient amounts of
the minor a-anomer for the NMR experiments, as being due to the
12.7
Average
1.023+0.006
b-Glucopyranoside 8
5
6
7
8
1.2
1.2
2.5
1.2
16.2
7.1
12.5
12.4
1:0.5
1:0.7
1:1.7
1:1
1.017
1.020
1.019
1.019
Average
1.019+0.001
*All KIEs were measured at –72 8C and were converted to 25 8C assuming
KIE258C ¼ exp{(201/298)ln(KIE272 8C)} (refs 27,29). †Errors quoted in the average values are at 1s
(see Supplementary Tables S1–S4 for details).
664
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1404
ARTICLES
a
c
b
d
13C KIE = 1.023; 2H KIE = 1.12
ΔG‡ = 26.2 kcal mol–1
13C KIE = 1.018; 2H KIE = 1.14
ΔG‡ = 19.3 kcal mol–1
13C KIE = 1.025; 2H KIE = 1.12
13C KIE = 1.011; 2H KIE = 1.20
ΔG‡ = 25.1 kcal mol–1
ΔG‡ = 19.5 kcal mol–1
r(C–OiPr) = 2.497 Å; r(C–OTf) = 2.423 Å
4H3
r(C–OiPr) = 2.430 Å; r(C–OTf) = 2.272 Å
B2,5
r(C–OiPr) = 2.423 Å; r(C–OTf) = 2.422 Å r(C–OiPr) = 2.527 Å; r(C–OTf) = 2.364 Å
4H3
B2,5
Figure 1 | Calculated (B3LYP) associative transition states for the reaction of isopropanol with 4,6-O-benzylidene protected manno- and glucopyransyl
triflates, leading to the formation of the a- and b-glycosides in each case. a–d, Transition states for the formation of a-mannoside (a), b-mannoside
(b), a-glucoside (c) and b-glucoside (d). For each transition state the calculated primary 13C and secondary 2H KIE values, the free energy of activation,
the lengths of the partial bonds to the leaving group and to the nucleophile, and the approximate conformation of the pyranose ring are listed.
separations corresponding to forming and breaking bonds, as well whereas those corresponding to the formation of the b-anomers
as the free energies of activation for each process. Additional (Fig. 1b,d) display nucleophile attachment ahead of leaving group
views, as well as other relevant structures, are provided in the com- departure. The differences in computed activation energies suggest
putational section of the Supplementary Information.
that the formation of b-mannoside 4 should be very strongly
The computed transition states in Fig. 1 represent what may favoured over a-anomer 3, whereas the same comparison for
loosely be termed concerted transition states for nucleophilic substi- glucose indicates the a-anomer 7 to be considerably favoured over
tution reactions in which the departing trifluoromethanesulfonate b-isomer 8 (the same preference observed experimentally in these
anion retains some degree of association with the putative glycosyl systems on the preparative scale, and to a lesser extent in the
oxocarbenium ion. Although intrinsic reaction coordinate (IRC) present experiments owing to the necessary use of both excess
calculations on each of these stationary points confirms that the Tf2O and limiting isopropanol—conditions that erode the selectiv-
single imaginary vibrational mode corresponds to nucleophile ity due to suppression of the selective ‘associative’ mechanism rela-
attachment to, and leaving group departure from, the anomeric tive to the competing unselective ‘dissociative’ mechanism21,22).
carbon (see Supplementary Information for IRC data, Figs S3–S10), Importantly, the computed 13C KIEs correspond quite well with
the differing degrees of C–OTf bond cleavage and C–OiPr the experimental values in these two cases (Fig. 1b,c), that is,
bond formation indicate different relative positions along the reac- 1.018 versus 1.023 for
4 and 1.025 versus 1.023 for 7.
2
tion coordinate. The transition states for the formation of the a-gly- Furthermore, the calculated secondary H KIE of 1.14 for the for-
cosides (Fig. 1a,c) are highly symmetrical, in that the bond to the mation of the b-mannoside 4 is in good agreement with the value
incoming alcohol is similar in length to that of the departing triflate, of 1.12 measured previously30.
_
TfO
Ph
O
Ph
O
+
+
+
O
Ph
O
Ph
O
O
Ph
O
O
O
O
O
O
_
OTf
O
O
O
MeO
MeO
TfO
MeO
MeO
MeO
_
MeO
MeO
OTf
TfO
MeO
MeO
MeO
β-CIP
α-Triflate
β-Triflate
SSIP (or free ions)
α-CIP
β-Mannoside and
β-Glucoside formation
ROH
ROH
α-Glucoside formation
Ph
O
Ph
O
O
ROH
α-Mannoside formation
O
O
O
OR
MeO
MeO
MeO
MeO
OR
β-Glycoside
α-Glycoside
Figure 2 | Mechanistic picture for the 4,6-O-benzylidene-directed formation of a- and b-gluco- and mannopyranosides. At the extreme left and right, a
series of equilibria connect two covalent glycosyl triflates with the corresponding CIPs and the more loosely associated SSIPs. In three of four cases studied,
reaction occurs in the ‘grey area’ defined by the equilibria between the covalent triflates and the CIPs. Formation of a-mannoside 3, however, clearly occurs
by a dissociative mechanism.
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1404
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The formation of the a-glucoside 7 through an apparent SN2-like coordinate; clearly, further work is necessary to probe the influence
transition state deserves comment as experimentally only the a- of acceptor–donor hydrogen bonding in these reactions.
glucosyl triflate 6 has been observed21–23, the implication being
Conclusion
that a kinetic scheme is in operation in which the more stable a-glu-
cosyl triflate is in rapid equilibrium with its less stable but more
reactive b-isomer, or its functional equivalent, a b-contact ion
pair (b-CIP). This type of kinetic scheme in which the less stable
of two isomers in very rapid equilibrium on the reaction timescale
is the more reactive one, leading to a situation in which the
product distribution bears no relation to the ratio of starting
materials, is a manifestation of one of the two boundary conditions
of the Curtin–Hammett principle36. The glaringly inconsistent
result is that obtained for formation of the a-mannoside (Fig. 1a),
where the computed 13C KIE of 1.023 differs significantly from
the average experimental value of 1.005 (Table 1). The implication
is that a-mannoside formation does not proceed through the com-
puted SN2-like transition state of Fig. 1a, but instead takes place at
the other end of the mechanistic spectrum through a dissociative
process that at least approaches the intermediacy of a distinct oxo-
carbenium ion and a triflate anion. The inability of computational
methods to locate such transition states16 without resort to artefact
such as the inclusion of a further cation to neutralize the charge on
the anion (owing to barrierless internal collapse) is widely appreci-
ated. This picture is fully consistent with the fact that the anomeric
effect is significantly smaller in the glucopyranosides than in the
mannopyranosides12,37, from which it follows that the Curtin–
Hammett scenario involving a minor b-triflate or a closely related
CIP is more likely in the gluco- than in the manno-series. The
recent demonstration of the existence of b-glycosyl triflates as
stable species when the substitution pattern is favourable further
supports the possibility of their intervention via a Curtin–
Hammett kinetic scheme38.
In summary, the agreement between computed and experimentally
determined primary 13C KIE values strongly suggests that the 4,6-O-
benzylidene directed b-mannosylation, as well as the b- and a-
glucosylation reactions, proceed through loosely associative
transition states in which the incoming nucleophile displaces the
leaving group from a loosely bound covalent glycosyl triflate of
the opposite configuration. In the case of a-glucoside formation,
the corollary of this observation is the invocation of a covalent b-
glucosyl triflate, or of a b-CIP, and the operation of Curtin–
Hammett-type kinetics. The contrast between the computed KIE
for the formation of the a-mannoside by a concerted pathway
and the experimentally observed value strongly suggests a highly
dissociative mechanism for this one example, which approaches
the intermediacy of a discrete glycosyl oxocarbenium ion. The dem-
onstration of associative transition states for three of the four reac-
tions studied suggests that the concentrations of both glycosyl
acceptor and donor may well have an impact on the stereoselectivity
of other glycosylation reactions in solution and points the way to the
more rational optimization of reaction conditions. By the same
token, the stereoselectivities of concentration-dependent glycosyla-
tions cannot be expected to translate directly to reactions conducted
on polymeric or other supports.
Methods
The 13C NMR spectra for the KIE measurements were recorded at 200 MHz using a
908 pulse, an inter-scan delay of 30 s, and 512 scans so as to meet the criterion of the
minimum 200:1 signal-to-noise ratio necessary for the precise integrations required.
Calculations were carried out using the Gaussian-09 suite of programs, with the
results visualized using GaussView 4.0 or 5.0 (ref. 41). All preparative, spectroscopic
and computational methods are reported in the Supplementary Information
together with full characterization data for all new compounds.
Overall, a picture emerges (Fig. 2) featuring a series of equilibria
encompassing, at the extremes, two covalent glycosyl triflates, the
corresponding CIPs and the more loosely associated solvent separ-
ated ion pairs (SSIP). For three of the four cases studied, the glyco-
sylation reaction has SN2 character, but as the KIE is at the lower end
of the expected value for such a concerted process the transition
states can be considered to be ‘exploded’ and thus possibly in the
grey area defined by reaction on a CIP. For this reason, the arrows
depicting the formation of the two b-products and the a-glucoside
Received 31 October 2011; accepted 7 June 2012;
published online 22 July 2012
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Acknowledgements
The authors acknowledge the Natural Sciences and Engineering Research Council
(NSERC) of Canada and the National Institutes of Health (NIH, GM62160) for grant
`
support to D.A.P. and D.C., respectively. M.H. and G.E.G. thank the Ministere de
l’Education Nationale de la Recherche et de la Technologie and NSERC, respectively, for
scholarships. D.A.P. acknowledges support from the Canada Research Chairs programme.
Author contributions
L.B., D.A.P. and D.C. designed the project and wrote the manuscript. M.H. carried out
the synthetic work. G.E.G. conducted the computational studies. N.B. and M.H. performed
the NMR study. M.H., N.B., L.B. and D.C. analysed the NMR studies, and G.E.G. and
D.A.P. analysed the computational work. All authors discussed the results and commented
on the manuscript.
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The authors declare no competing financial interests. Supplementary information and
31. Crich, D., Smith, M., Yao, Q. & Picione, J. 2,4,6-Tri-tert-butylpyrimidine
(TTBP): a cost effective, readily available alternative to the hindered base
2,6-di-tert-butylpyridine and its 4-substituted derivatives in glycosylation
and other reactions. Synthesis 323–326 (2001).
chemical compound information accompany this paper at www.nature.com/
naturechemistry. Reprints and permission information is available online at http://www.
nature.com/reprints. Correspondence and requests for materials should be addressed to
D.A.P. and D.C.
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