Native Serine Peptide Assembly – Scope and Utility

Article abstract of DOI:10.1002/ejoc.201601148

This work develops serine peptide assembly (SPA), which complements and contrasts with classic native chemical ligation (NCL). Advances in reagentless peptide-bond formation have been applied to serine (and serine models) and a range of C-terminal amino acids, including bulky residues that are not amenable to NCL. The particular appeal of SPA is preparative-scale segment condensations with zero racemization risk and favorable process mass intensity (PMI). Mechanistic studies support a previously proposed reaction pathway via an initial transesterification step. An understanding of the factors favoring this pathway relies on hard–soft acid–base theory, according to which mildly activated esters with the largest carbonyl positive charge are most reactive with hydroxyamines. Novel C-terminal activators have been discovered that enhance reactivity and give harmless byproducts.

Full text of DOI:10.1002/ejoc.201601148

DOI: 10.1002/ejoc.201601148
Communication
Hydroxyamine Amidation
Native Serine Peptide Assembly – Scope and Utility
[a]
[a]
Michael C. Pirrung* and Ryan S. Schreihans
Abstract: This work develops serine peptide assembly (SPA), port a previously proposed reaction pathway via an initial trans-
which complements and contrasts with classic native chemical esterification step. An understanding of the factors favoring this
ligation (NCL). Advances in reagentless peptide-bond formation pathway relies on hard–soft acid–base theory, according to
have been applied to serine (and serine models) and a range which mildly activated esters with the largest carbonyl positive
of C-terminal amino acids, including bulky residues that are not charge are most reactive with hydroxyamines. Novel C-terminal
amenable to NCL. The particular appeal of SPA is preparative- activators have been discovered that enhance reactivity and
scale segment condensations with zero racemization risk and give harmless byproducts.
favorable process mass intensity (PMI). Mechanistic studies sup-
Introduction
formed in relatively nonpolar media at ambient temperature for
extended periods or more rapidly with microwave heating.
We recently introduced a method for reagentless amide bond
formation with a focused set of amines, those bearing nearby
[
1]
hydroxy groups. Their carboxyl reaction partners were esters Results and Discussion
mildly activated for acyl transfer by strain or inductive effects
To study a reaction partner that better represents the C-termi-
or both. We suggested that the pathway for these reactions
involves initial transesterification of the alcohol and ensuing re-
arrangement to give the more stable hydroxyamide. Envision-
ing the application of this technology to preparative peptide
segment condensations at N-terminal serine residues, we aim
here to expand the scope to diverse activated acyl derivatives,
including native amino acids with various mildly activated es-
ters. This work provides further support for the transesterifica-
tion/rearrangement pathway that mimics native chemical liga-
tion (NCL) and has uncovered readily introduced, superior C-
terminal activating groups.
nus of a peptide segment, we replaced the carbamate N-protec-
tion from the earlier study with an N-acetyl group. Particularly,
N-acyl amino acids (like peptide C-termini) are vulnerable to
racemization when the carboxylate is activated with conven-
tional coupling agents, because of competing oxazolone forma-
tion that labilizes the α-hydrogen of the C-terminal residue. In
examining the relative reactivity of different native amino acids,
L-alaninol was used as a simple serine surrogate. Each of the
known acetyl amino acids in Table 1 was converted to its cyano-
methyl (CM) ester 3 in 80–95 % yield under the conditions re-
ported earlier: 1 equiv. Cs CO , 1.5 equiv. BrCH CN, ambient
2
3
2
Our earlier work was limited to N-Boc-valine. The valine carb-
oxylate was activated as a cyanomethyl ester or oxazolidinone
temperature, 1.5–8 h. These esters were allowed to react with
a 50 % excess amount of alaninol, either at ambient tempera-
(
1, Scheme 1). Nonetheless, successful amide formation with
ture or with microwave heating at 100 °C, in 1
M solutions. Ethyl
this amino acid derivative (with its bulky α-substituent) far sur-
passes the capabilities of NCL, which is the sulfur relative of
[
2]
Table 1. Peptide-bond formation with N-acetylamino cyanomethyl esters.
SPA. In NCL, valine is tolerated at the C-terminus only when
[
3]
using a selenoester with selenocysteine. Assemblies were per-
Entry
Ac-AA
Time [h][a]
Yield [%] Time [min][b] Yield [%]
a
b
c
d
e
f
Val
Leu
Phe
Met
Pro
Gly
(Trt)Asn
(Bn)Cys
72
72
48
48
72
72
24
48
78
38
76
90
64
72
79
78
90
90
90
90
90
90
ND[
ND
78
53
76
91
54
73
ND
ND
Scheme 1. Initial peptide assembly with serine.
[
a] Department of Chemistry, University of California,
Riverside, CA 92521 USA
c]
g
h
E-mail: Michael.pirrung@ucr.edu
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/ejoc.201601148.
[a] Ambient temperature. [b] Microwave heating. [c] ND: not determined.
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Communication
acetate proved a useful solvent for these reactions, dissolving
The N-acetyl oxazolidinones 6 of several native amino acids
polar reactants that are insoluble in the hydrocarbons that were were also investigated. These are prepared from the N-acetyl
preferred earlier, but still providing a relatively nonpolar me- amino acids in 55–84 % yield by treatment with pTsA and para-
dium that gives higher conversions. The results are summarized formaldehyde in toluene for 3–12 h. As Table 2 shows, the
in Table 1.
trends observed in the CM ester study hold here, the Leu deriv-
Table 1 shows that significant variation in the C-terminal ative again being lower-yielding.
amino acid is very well-tolerated. Leucine is a slower reactant,
Table 2. Peptide-bond formation with N-acetyl oxazolidinones.
which can be explained on the basis of syn-pentane interac-
[
4]
tions or other remote steric effects. Successful reaction with
[
5]
proline is notable, as Pro (like Val) is not useful in NCL. Though
[
6]
some advancements have been made in this area, C-terminal
bulk is still a significant problem for NCL. No racemization (less
than 0.1 %) was observed in any of these reactions, as estab-
lished by detailed spectroscopic examination of the products in
Entry
Ac-AA
Time [h][a]
Yield [%] Time [min][b] Yield [%]
comparison with the peptide diastereomer generated with
alaninol.
D-
a
b
c
Val
72
72
48
36
81
41
78
82
90
90
90
90
78
58
74
76
Leu
Phe
Met
We also aimed to provide greater support for the mechanism
postulated earlier; that is, an initial transesterification, enhanced
by internal H-bonding between the basic amine and the
hydroxyl group, followed by a rearrangement (via transacyl-
ation) to produce the hydroxyamide. Structural variations were
made to the amino alcohol to examine this issue. As Table 1
shows, when Ac-Val-OCM reacts with alaninol under ambient
conditions, the formation of 4a is complete in 72 h. When N,N-
dimethylalaninol is substituted, the transesterification product
d
[a] Ambient. [b] Microwave heating.
When extending these reactions of 3a or 6a to SPA with
ethyl serinate, a reactant more representative of a peptide N-
terminus, yields were only moderate. This stimulated the search
for superior C-terminal mildly activated esters that could be
readily generated from peptides. Boc-Val was used as a test
amino acid as it maintains the steric demand uniquely tolerated
in hydroxy amine amidation while enabling the rapid synthesis
of a wide range of esters using multiple conventional methods.
This study was performed by treating valine esters 7 with
alaninol under our standard ambient reaction conditions and
observing the time for complete consumption of starting mate-
rial to form 8. The results in Table 3 show interesting features.
Although CM esters have been used in transesterification reac-
5
is formed in 89 % yield, also in 72 h. When alaninol N-form-
amide is used, there is no reaction. When alaninol tert-butyldi-
methylsilyl ether is used, there is no reaction (Scheme 2). We
showed earlier that alaninol tert-butyldimethylsilyl ether does
not react with a γ-lactone, demonstrating the essential nature
[
1]
of the alcohol in these amide-forming reactions. The current
results show that transesterification is kinetically competent as
the initial step in the overall process, that direct acylation is
not a favorable reaction pathway, and that transesterification
requires a nearby basic nitrogen. All of these data support the
mechanism discussed above, as explicitly detailed in Scheme 3.
[
7]
tions in the past, they are among the slowest of all investi-
gated reactive esters. A significant rate gain is observed with
fluorinated esters, the best being the vinyl esters. The fastest,
(methyl trifluorocrotonate)yl (entry m), is prepared by conjugate
[
8]
addition of the carboxylate to ethyl trifluorobutynoate. Hexa-
Table 3. Boc-Val-ester reactivity under ambient conditions.
Entry
R
Time [h]
a
b
c
d
e
f
g
h
i
CH
CH
CH
CH
CH
CH
CH
CH
CH
2
2
2
2
2
2
2
2
2
SCH
CCl
3
84
80
76
70
54
50
48
32
28
18
18
15
8
3
CH Cl
2
Scheme 2. Results supporting the transesterification/rearrangement pathway.
CN
CHCl
SOCH
SO CH
2
3
2
3
CF
CF
3
2
CF
3
j
k
l
CH(CF
C=CH
(MeO
(CF )C=CH(CO
2
3 2
)
2
[C(CF
C)C=CH(CO
Me)
3
)
2
CF
2
CF
3
]
2
2
Me)
m
3
Scheme 3. The transesterification/rearrangement mechanism.
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2
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Communication
fluoroisopropyl (HIP) and trifluoroethyl (TFE) esters also have
As many chemists would judge nitrogen as intrinsically more
good reactivity. Some other Boc-Val esters (structures in the nucleophilic than oxygen, the mechanistic pathway inferred for
Supporting Information) show essentially no reaction, despite a SPA is counterintuitive. Likewise, it could be said that sulfur is
reasonable expectation of good reactivity based on their group more nucleophilic than either, hence its utility in NCL. However,
electronegativities (vide infra).
it is known that oxy anions are more nucleophilic than amide
Preparative yields for the six most reactive esters 7 for SPA anions, as shown by Brønsted linear free energy relationships
[
9]
with ethyl serinate (Scheme 4) were determined under the stan- in substitution reactions (on benzyl chloride). The accessibility
dard reaction conditions. Additional products that diminished of the oxy anion is also generally better, owing to intrinsically
the isolated yields of Boc-Val-Ser-OEt (2) were observed with lower pK
the three vinyl esters, but the HIP and TFE esters gave yields reactants based on the internal basic amine.
s for alcohols than for amines, and specifically in these
A
greater than 92 % in 20–32 h under ambient conditions. Experi-
In considering these results, we aimed to develop structure–
ments to establish the reaction pathway for amide formation reactivity correlations that would enable understanding and
were performed with 7j to parallel those with 3a (Scheme 2), prediction of ester reactivity in SPA (see tables in the Support-
with the same outcome: the transesterification product is ing Information). Group electronegativity data is available for
formed from N,N-dimethylalaninol in 94 % yield in the same some of the ester groups used, but it shows poor agreement
reaction time, which means that this step is kinetically compe- with reactivity. For example, the ethynyl and cyano groups have
[
10]
tent, and no other alaninol analogs react.
the same group electronegativity (3.3),
but propargyl esters
are unreactive whereas cyanomethyl esters react fairly well.
Other correlations were based on electronic structure calcula-
tions using the PM3 semiempirical method. The LUMO energies
and carbonyl electrostatic charges were examined for acetyl de-
rivatives of ester groups from Table 3, as well as other com-
monly used active esters. The best predictor of the observed
reactivity (with alaninol) was the electrostatic charge on the
Scheme 4. Preparation of 2 from the six most reactive esters 7.
Identification of these superior esters prompted develop- carbonyl carbon atom. We interpret this preference as the tend-
ment of methods for mild ester formation from free C-termini ency of the hard nucleophile alkoxide to transesterify faster
by using N-Ac-Val as a model. In Sn2 reactions with the carb- with harder carbonyls.
oxylate as the nucleophile, commercially available trifluoroethyl
triflate proved about as reactive in forming TFE esters as the tainability of chemical processes, a prominent measure is proc-
Of the metrics that have been proposed to evaluate the sus-
[
11]
bromoacetonitrile used to make CM esters, providing the ester ess mass intensity (PMI).
This is the ratio of the mass of all
in essentially quantitative crude yield in a few hours. However, materials entering a reaction (excluding aqueous solvents) to
the TFE ester is more desirable, because it is more reactive and the mass of final product, the best possible PMI being 1. For
its byproduct is volatile and nontoxic (Scheme 5). Like CM es- the preparation of tripeptides 10 and 12 by SPA, the two-step
ters, mildly activated TFE esters are formed without generating PMI is approximately 5. This is a relatively low value and con-
a reactive acylating agent from the carboxyl group, which elimi- trasts with higher PMI values observed for conventional peptide
nates all concerns about racemization via oxazolone formation. couplings that include condensing agents and additives to sup-
These reactions have been performed on scales up to 1 g of press racemizations that are unnecessary in SPA.
TFE ester 9. It is stable upon storage and has proved resistant
to racemization even with microwave heating in ethyl acetate
under reaction conditions in the absence of a hydroxyamine. Conclusion
We have generally observed that these mildly activated esters
This work complements the substantial achievements in liga-
can be used in 20–50 % excess and that the unreacted starting
tions at Ser and Thr residues with unprotected polypeptides in
material can be recovered after the reaction and reused.
[
12]
dilute aqueous media.
Experimental Section
Ac-Val-OTFE (9): Cesium carbonate (1.0 mmol, 325.3 mg) was
added to a solution of N-Ac-Val (1.0 mmol, 217.3 mg) in acetonitrile
(
10 mL). This solution was stirred at room temperature for 15 min,
and then 2,2,2-trifluoroethyl trifluoromethanesulfonate (1.5 mmol,
48.2 mg) was added in one portion. The reaction mixture was
Scheme 5. Model tripeptide syntheses using SPA of TFE esters.
3
We applied serine peptide assembly to two simple examples. stirred at room temperature for 3 h and filtered through Celite. The
On treatment of 9 with seryl-phenylalanine ester, tripeptide 10 filtrate was concentrated in vacuo and purified with silica gel flash
chromatography using ethyl acetate as eluent to yield 228 mg
95 %) of the title compound as a colorless oil.
forms in 76 % yield in 36 h (room temp.). Changing the CM
ester to the TFE ester mitigated the earlier difficulty with leu-
(
cine, as treatment of 11 with seryl-phenylalanine ester forms Ac-Val-Ser-Phe-OMe (10): Ac-Val-OTFE (0.15 mmol, 36.6 mg) was
tripeptide 12 in 66 % yield (48 h, room temp.).
added to a solution of Ser-Phe-OMe (0.13 mmol, 33.6 mg) in ethyl
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Communication
acetate (0.13 mL). The reaction mixture was stirred at room temper-
ature for 36 h and concentrated in vacuo. The residue was purified
by silica gel chromatography using 10 % methanol in dichloro-
methane as eluent to yield 48.7 mg (76 %) of the title compound
as a white solid. M.p. 202 °C (decomposes).
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Acknowledgments
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This work was partially supported by grants from National Sci-
ence Foundation (NSF) (CHE 1362737) and the National Insti-
tutes of Health (NIH) (AI106597). We are grateful to Kian Sedighi
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Keywords: Amides · Amino acids · Acylation · Nucleophilic
substitution · Transesterification
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2
Received: September 15, 2016
Published Online: ■
9
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Communication
Hydroxyamine Amidation
Surprisingly, mildly activated esters of
α-amino acids prefer to react with
amino alcohol nucleophiles to first
transesterify, guided by a favorable
hard–hard interaction of the alcohol
nucleophile and carbonyl positive
charge, and then undergo a dyotropic-
like rearrangement to form serine
peptide bonds.
M. C. Pirrung,* R. S. Schreihans ..... 1–5
Native Serine Peptide Assembly –
Scope and Utility
DOI: 10.1002/ejoc.201601148
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim