Curtin-Hammett and steric effects in HOBt acylation regiochemistry

Article abstract of DOI:10.1021/jo200346r

While hydroxybenzotriazole is commonly used in a variety of bond-forming reactions, its acylation has been shown to produce a regiochemical (O vs N) mixture with complex kinetic behavior. Increased steric bulk on the electrophile favors formation of the o

Full text of DOI:10.1021/jo200346r

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CurtinꢀHammett and Steric Effects in HOBt Acylation Regiochemistry
Benjamin D. Brink, Justin R. DeFrancisco, Julie A. Hillner, and Brian R. Linton*
Department of Chemistry, College of the Holy Cross, One College Street, Worcester, Massachusetts 01610, United States
S Supporting Information
b
ABSTRACT: While hydroxybenzotriazole is commonly used
in a variety of bond-forming reactions, its acylation has been
shown to produce a regiochemical (O vs N) mixture with
complex kinetic behavior. Increased steric bulk on the electro-
phile favors formation of the oxygen-acylated product. Upon
standing as a solid, the mixture can isomerize completely to the
nitrogen adduct. An equilibrium ratio of regioisomers can be re-
established in solution by adding either nucleophilic or electro-
philic reagents, suggesting that the composition of the mixture
is not significant to subsequent reactivity. Solvents can affect this regiochemical equilibrium through a CurtinꢀHammett effect,
where the shift in the tautomeric equilibrium of HOBt in polar solvents biases the reaction toward the oxygen adduct.
’ INTRODUCTION
ethyldimethylaminopropylcarbodiimide (EDC) are shown in
Table 1. Remarkably nonuniform behavior was observed. Less
sterically hindered derivatives (such as Boc-glycine 2a) showed a
product ratio that favored the N-acyl product 4, but as the steric
bulk increased the product ratio shifted toward the O-acyl
derivative. This change was subtle for the methyl side chain of
alanine (2b), more pronounced in the isopropyl side chain of
valine (2c), and nearly complete in the case of the two methyl
groups of Aib (2d). In each case where two products were
observed, there was doubling of both the HOBt aromatic protons
as well as the aliphatic signals. A proportional steric dependence
of product ratios was observed for simple carboxylic acids as well
(2eꢀi). The accumulated data suggests that the N-acyl isomer 4
has greater steric limitations on its formation, and acylation with
bulkier substrates favors the formation of the O-acyl derivative 3
that contains an extra atom between the HOBt rings and the
acylating agent. The steric effect is consistent within a group of
similar molecules, but comparison of amino acids and simple
carboxylic acids suggests that this is not the only effect. These
regiochemical results are consistent with the scattered reports of
HOBt acylation existing in the literature, where both mixtures
and pure compounds have been reported. For example, when Aib
was chosen to probe the maximal steric deterrent for coupling
only the O-acyl form was observed,8 but less sterically demanding
substrates led to mixtures.5 One additional point of note is that
only one regioisomer was ever observed with acylated HOAt
derivatives under any reaction conditions and no isomerization
was observed, suggesting that these effects are not general to all
similar derivatives.9
A wide variety of coupling conditions have been developed
that promote the chemical synthesis of carboxylic derivatives.1
One method to minimize side reactions is to include a nucleo-
philic additive that forms an activated ester derivative and
subsequently reacts with the desired nucleophile. Some of these
additives include N-hydroxysuccinimide (HOSu),2 N-hydroxy-
benzotriazole (HOBt),3 and ethyl 2-cyano-2-(hydroxyimino)-
acetate (OXYMA).4 All of these additives can be added in the
course of a one-pot coupling reaction or the activated ester
derivative can be isolated. An effort to maximize synthetic yields
led us to isolate HOBt esters of various amino acid derivatives
(Figure 1). In several cases, instead of obtaining one clean HOBt
adduct, two regioisomers (3 and 4) were formed. Similar
isomeric results have been reported previously but are poorly
understood.5
HOBt 1 is unique among these coupling additives in that it can
form more than one acylated derivative. HOBt has been isolated
in two tautomeric forms6 and has been shown to be an ambident
nucleophile where both the oxygen and an aromatic nitrogen are
capable of forming bonds with electrophiles. This regiochemical
ambiguity has been seen in the structures of coupling reagents
that contain HOBt, such as HBTU,7 as well as a variety of
reactions that form isomeric HOBt adducts.5 It remains unclear
what factors control this regiochemical selectivity, whether
conditions can be modified to promote one isomer or the other
and how the shifting identity of the activated esters (3 and 4)
affects subsequent reactivity.
One aspect of these reactions that has not been reported
previously is the isomerization of 3 to 4 upon formation of a solid.
’ RESULTS AND DISCUSSION
The formation of a variety of acylated HOBt derivatives was
undertaken to assess the regiochemical ambiguity (Figure 1).
The resulting product ratios for a variety of amino acids (2aꢀd)
under coupling conditions with HOBt and the coupling reagent
Received: February 16, 2011
Published: May 23, 2011
r
2011 American Chemical Society
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Figure 1. Creation of acylated HOBt regiochemical isomers.
Table 1. Isolated Product Ratios for Reaction of Carboxylic
Acid Derivatives (2aꢀi) with HOBt and EDC
acylating agent
O-acyl 3/N-acyl 4
Boc-Gly-OH 2a
Boc-Ala-OH 2b
Boc-Val-OH 2c
Boc-Aib-OH 2d
MeCO2H 2e
EtCO2H 2f
25:75
42:58
70:30
97: 3
43:57
48:52
75:25
99:1
iPrCO2H 2g
tBuCO2H 2h
PhCO2H 2i
99:1
In the preliminary stage of the syntheses found in Table 1, it was
noticed that the product ratios were often dependent on the time
frame of the reaction and workup. Some purification conditions
and NMR acquisition led to the product ratios reported in the
table, while delays led to an increase in product 4. Indeed, when
samples that contained a mixture of 3 and 4 were allowed to stand
as a solid for a period of time, exclusively N-acyl product 4 was
often observed, without any signs of degradation products. As a
result, all of the data in Table 1 reflect immediate workup and
characterization. This isomerization will be addressed further in
the kinetics results below.
Kinetics. A more detailed kinetic analysis of HOBt acylation
was undertaken and proved to be quite enlightening in regard to
the regiochemical selectivity. The acylation of HOBt with
propionyl chloride was chosen as a characteristic reaction, and
the kinetic progress was monitored in various NMR solvents.
Figure 2 shows the percentages of the O-acyl isomer 3f in the
mixture of 3f and 4f in four solvents. In each case, the acyl
chloride is consumed before NMR acquisition is possible (within
3 min), and the O-acyl product is formed exclusively at early time
points. As each experiment progressed, an isomerization was
observed that led to the final equilibrium product ratios shown in
Table 2. While the reaction trends are the same in every solvent,
the final equilibrium product ratio was different in each solvent.
The reaction of propionyl chloride in CDCl3 showed a final
product ratio that was nearly identical to propionic acid under
coupling conditions, slightly favoring the N-acylation product 4f.
Moreover, this product ratio was unchanged by performing the
reaction at reflux, suggesting that an equilibrium mixture had
been achieved. When benzene, acetonitrile, or acetone was used
as the solvent the reaction favored O-acylation product 3f, each
to a different degree.10 Identical product ratios are observed
when triethylamine is used as a base, but the equilibrium is
reached at a much faster rate.11
Figure 2. Kinetics for the reaction of propionyl chloride and HOBt in
various solvents. 2 = CDCl3; O = C6D6; 9 = CD3CN; ] =
CD3C(O)CD3.
Table 2. Final Product Ratios for the HOBt Acylation with
Propionyl Chloride in NMR Solvents
reaction solvent
O-acyl 3f:N-acyl 4f
CDCl3
47:53
47:53
58:42
78:22
84:16
CDCl3 at reflux
C6D6
CD3CN
CD3C(O)CD3
reflect a balance of tautomeric equilibrium, relative nucleophili-
city, and product stability. HOBt has been shown to exist in two
tautomeric forms (1 and 10 Figure 3) with more polar solvents
biasing this equilibrium toward the zwitterionic form.6 The initial
formation of the O-acyl derivative 3 in all solvents suggests that
the reaction at the oxygen atom proceeds through a lower
activation energy than the ring nitrogen, presumably due to a
more accessible steric approach. The ultimate formation of N-
acyl product 4 upon formation of a solid suggests this is the
thermodynamic product.13 The greater stability of the nitrogen/
carbonyl bond over an oxygen/carbonyl bond appears to be
similar to the relative stability of other amide and ester deriva-
tives. Presumably over time, molecules in equilibrium can over-
come the higher barrier to form N-acyl adduct 4. There appear to
be two competing solvent effects. The shift toward the O-acyl
product 3 in benzene in comparison with chloroform is con-
sistent with a disfavoring of the zwitterionic N-acyl product 4 in
the more nonpolar conditions. An alternative effect is seen with
acetone and acetonitrile as solvent, where presumably the more
This equilibrium behavior appears to be an example of the
CurtinꢀHammett principle,12 where the results in various solvents
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Figure 3. CurtinꢀHammett equilibrium of HOBt tautomers and acylated HOBt adducts.
Figure 4. Regiochemical mixture and enriched isomers from the reaction of propionyl chloride and HOBt: (a) reaction mixture in CDCl3; (b) N-acyl
form 4f after several days as a solid; (c) enriched O-acyl form 3f (95% purity) isolated from immediate workup.
polar product should be favored. The observed preference for
O-acylation correlates with the change in relative tautomeriza-
tion of the HOBt nucleophile in solution during the course of the
reaction, with more polar solvents stabilizing the zwitterionic
tautomer 10 and shifting the equilibrium toward the O-acyl
isomer 3.14 This is the example of the CurtinꢀHammett
principle where the balance of tautomers or transition states
exhibits a greater influence on the reaction than does product
stability.
These syntheses and kinetic profiles illustrated methods to
form each regioisomer in relative purity (Figure 4). The O-acyl
derivative 3 could be isolated from short reaction timeframes
without added base, followed by immediate extraction with
water.15 In this manner, the O-acyl isomer could be obtained
in 95% purity. The N-acyl derivative 4 could be obtained merely
by letting the compound stand in the solid state for a matter of
Figure 5. Re-equilibration of mixtures of 3f and 4f to the same 47:53
ratio. The percentage of O-acyl 3f in the 3f/4f mixture are shown. All
days, and complete isomerization was observed for simple
carboxylic acids. Conversely, when a mixture of 3 and 4 was
reactions are in CDCl3. 2 = propionyl chloride þ HOBt (2 equiv); b =
retained in CDCl3 solution, the product ratio was unchanged
N-acyl derivative 4f þ DMAP; O = enriched O-acyl derivative 3f þ
DMAP; [ = N-acyl derivative 4f þ HOBt; 9 = enriched N-acyl
derivative 4f þ EtCOCl; 0 = enriched O-acyl derivative 3f þ EtCOCl.
over a period of three weeks.10 This was found to be the case
independent of the starting ratio itself. These results suggest that
once the purified product ratio is established it is maintained in
solution, but the formation of the more stable N-acyl 4 product is
favored in the solid.
dissolved in CDCl3 in the presence of DMAP, the 47:53 equili-
brium ratio was almost immediately reestablished (Figure 5, b).
Similar behavior was observed when DMAP was added to a
mixture enriched in the O-acyl derivative 3f as well (Figure 5, O).
This would suggest that the DMAP functions as a nucleophilic
While any existing ratio of 3f and 4f was unchanged in pure
solvent, additional nucleophilic or electrophilic reagents led to an
isomerization back to the equilibrium ratio reported in Figure 2
(see Figure 5, 2). When the pure N-acyl derivative 4f was
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catalyst as shown in Figure 6a to form 5, liberating HOBt which
can subsequently react to re-establish an equilibrium of 3f and 4f.
Addition of HOBt itself also led to the re-equilibration to the
47:53 ratio at a much slower rate (Figure 5, (). A similar slower
re-equilibration was observed when additional acyl chloride was
added to CDCl3 solutions enriched in either regioisomer
(Figure 5 9, 0). All kinetic curves converge on the equilibrium
ratio of 47:53 given enough time.10 It is more difficult to suggest
exactly how the acyl chloride accelerates the rate over solvent
alone, but the excess of acyl halide would preclude the liberation
of HOBt. One possibility is shown in Figure 6b, where the
acylation of the HOBt oxygen atom of the N-acyl adduct 4,
followed by breakdown of the bis-acylated HOBt adduct 6.16
When similar experiments with added nucleophile were per-
formed in other solvents, the re-equilibration reached values
similar to the data in Table 2.10
to nucleophilic attack than the N-acyl isomer 4.5 In our hands,
when a mixture of 3f and 4f was reacted with butylamine, both
isomers were consumed equally. This is complicated, however,
by the re-equilibrations described above. When a solution of pure
N-acyl isomer 4f was reacted with a substoichiometric amount of
amine, the O-acyl isomer 3f was formed as well as the product
amide. Presumably, the nucleophilic attack liberated HOBt
which subsequently re-established the equilibrium ratio of 3
and 4. This competing process makes it impossible to determine
if both isomers are indeed reacting equally, or if one isomer reacts
followed by re-establishment of the equilibrium ratio. One
exception was the reaction of an equilibrium mixture of 3f and
4f in CDCl3 at 0 °C with substoichiometric tert-butylamine. In
this case, some N-acyl isomer 4f remained in solution and the O-
acyl isomer 3f was completely consumed, while HOBt precipi-
tated from solution. It is thought that the more electrophilic O-
acyl derivative reacted preferentially and precipitation of HOBt
from the reaction at low temperature precluded re-equilibration.
In general, the O-acyl isomer does appear to be more electro-
philic, but liberation of HOBt can result in the subsequent
formation of an equilibrium mixture of both regioisomers. This
would mean that in many cases the composition of the acylated
mixture would have little effect on subsequent reactivity.
Reactivity toward Nucleophilic Attack. Previous results
have suggested that the O-acyl derivative 3 can be more reactive
Formation of N-Acyl Isomer in Solid. The ultimate isomer-
ization to the N-acyl isomer 4 upon isolation of the pure solid is
more challenging to address mechanistically. Figure 7 shows
parallel reactions that follow the progress of a sample containing
both 3f and 4f, one portion being kept in CDCl3 solution while
the other portion was evaporated to a solid. The solution again
remained unchanged, while the solid ultimately isomerized to the
N-acyl form 4f through a complex kinetic profile, without the
formation of other products. The results in Figures 2 and 5
suggest that if any reaction occurs in solution it will lead to the
establishment of an equilibrium of both acylated-HOBt regioi-
somers, with nucleophilic catalysis occurring fairly quickly and
electrophilic catalysis occurring more slowly. Neither of these
processes occurs at any appreciable rate in solution in the absence
of catalyst, with an existing product ratio being stable for a matter
of weeks. The ultimate establishment of the thermodynamic
product in the solid requires both the cleavage of existing bonds,
and reformation of the other isomer without degradation. One
could imagine this resulting through either a greater shift toward
nitrogen nucleophilicity in the absence of solvent, or through a
process that is not observed in solution. There is no NMR
evidence of hydrolysis to liberate HOBt to subsequently function
as a nitrogen nucleophile and no solvent polarity where reaction
at nitrogen is exclusive (Table 2). One possibility is that a
bimolecular mechanism would become more favorable at higher
concentrations, such as in a solid. The proposed mechanism in
Figure 8 shows a bimolecular electrophilic process with the more
electrophilic O-acyl substrate reacting with another molecule of
Figure 6. Potential mechanisms for the re-equilibration of N-acyl HOBt
through (a) nucleophilic catalysis or (b) electrophilic catalysis.
Figure 7. Kinetic profile for two sample containing both 3f and 4f. [ =
solution in CDCl3; 9 = evaporated to a solid.
Figure 8. Possible mechanism for the isomerization to 4 observed in the solid state.
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