5-Methylisoxazole-3-carboxamide-Directed Palladium-Catalyzed γ-C(sp3)-H Acetoxylation and Application to the Synthesis of γ-Mercapto Amino Acids for Native Chemical Ligation

Article abstract of DOI:10.1021/acs.orglett.6b01160

Palladium-catalyzed acetoxylation of the primary γ-C(sp³)-H bonds in the amino acids Val, Thr, and Ile was achieved using a newly discovered 5-methylisoxazole-3-carboxamide directing group. The γ-acetoxylated α-amino acid derivatives could be e

Full text of DOI:10.1021/acs.orglett.6b01160

Letter
pubs.acs.org/OrgLett
5‑Methylisoxazole-3-carboxamide-Directed Palladium-Catalyzed
γ‑C(sp3)−H Acetoxylation and Application to the Synthesis of
γ‑Mercapto Amino Acids for Native Chemical Ligation
Kalyan Kumar Pasunooti, Renliang Yang, Biplab Banerjee, Terence Yap, and Chuan-Fa Liu*
School of Biological Sciences, Nanyang Technological University, Singapore 637551
S
* Supporting Information
ABSTRACT: Palladium-catalyzed acetoxylation of the primary γ-C(sp3)−H bonds in the amino acids Val, Thr, and Ile was
achieved using a newly discovered 5-methylisoxazole-3-carboxamide directing group. The γ-acetoxylated α-amino acid derivatives
could be easily converted to γ-mercapto amino acids, which are useful for native chemical ligation (NCL). The first application of
NCL at isoleucine in the semisynthesis of a Xenopus histone H3 protein was also demonstrated.
ecent years have witnessed tremendous advances in the
development of methodologies for selective functionaliza-
amino acids would be via direct functionalization of their
natural amino acid counterparts; however, so far this has been
realized only for amino acids with “activated” C−H bonds on
the side chain,8a,m,n,p,q,s such as Asp and Glu of which the side-
chain COOH when in its ester form makes the respective β and
γ-C−H bonds more acidic for direct functionalization.8m,n,p For
example, a β-HS-Asp analogue can be synthesized stereo-
selectively in three steps,8m but this cannot work for
“inactivated systems” in amino acids with inert β and γ-C−H
bonds. For these systems, the aforementioned transition-metal
catalyzed C−H activation presents a conceptually attractive way
to access their thiol-modified derivatives. Figure 1 shows such a
synthetic strategy for certain γ-mercapto-α-amino acids.
R
tion of inert C(sp3)−H bonds using transition-metal catalysts.1
The success is largely attributed to the use of the so-called
directing groups or auxiliaries, which can bond and deliver the
metal atom to a particular C−H bond for its activation via
cyclometalation.2 The transformational simplicity of direct
C(sp3)−H functionalization endows it with great potential in
organic synthesis, and recent advances have helped to establish
it as a viable methodology in the synthesis of complex organic
compounds such as natural products and pharmaceutics.3
Another interesting application is the synthesis of certain
nonproteinogenic “unnatural” amino acids through the direct
functionalization of C(sp3)−H bonds in simple amino acids.4,5
Among the many uses of unnatural amino acids, one is ascribed
to native chemical ligation (NCL), which in its original form
utilizes an N-terminal Cys residue of one peptide to mediate
ligation with another thioester peptide to form an Xaa-Cys
peptide bond at the ligation junction.6 As an extension of NCL,
many unnatural β or γ-mercapto amino acids have been
prepared and used as NCL mediators for ligation at non-Cys
junctions through a ligation−desulfurization strategy which was
first introduced by Dawson et al.7,8 Despite the great utility of
these mercapto amino acids for NCL, their synthesis using
existing methods is often very tedious and inefficient due to
ramifications in the steps of carbon framework construction,
introduction of functional groups, and protection and
deprotection maneuvers.9 New and more efficient methods to
prepare these mercaptoamino acids continue to be well sought
after. An attractive route to the synthesis of these mercapto
Sanford10 and Yu11 demonstrated early examples of Pd-
catalyzed selective oxygenation of inert C(sp3)−H bonds in
simple organic compounds using monodentate nitrogen-based
Figure 1. Pd-catalyzed direct acetoxylation of γ-CH3-containing α-
amino acid derivatives and conversion to γ-mercapto amino acids. DG
= directing group.
Received: April 21, 2016
© XXXX American Chemical Society
A
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Letter
directing groups. Based on the pioneering work of Daugulis on
removable bidentate 8-aminoquinoline (AQ) and picolinamide
(PA) directing groups for C−H activation,12 Corey first showed
AQ-directed β-acetoxylation in amino acid substrates.13
Subsequently, both AQ and PA as well as other directing
groups have been used for the β and γ-acetoxylation of
carboxylic acid and alkylamine substrates including some amino
acids.14 We realized that such a method for inert C−H
acetoxylation would be useful to prepare the otherwise difficult-
to-prepare γ-mercaptoamino acids. As the reaction occurs on
the γ-C, it would avoid the challenges involved in obtaining the
desired chiral configuration at the β-carbon of threonine or
isoleucine when using conventional synthetic methods. γ-
Mercaptoisoleucine would be a particularly difficult target for
the traditional methods, and likely for this reason it has not
been reported thus far for use in NCL.
Herein, we show that our newly discovered 5-methyl
isoxazole-3-carboxamide (MICA) auxiliary group15 can be
used to direct palladium-catalyzed C−H acetoxylation on the
γ-carbon of Val, Thr, and Ile. Moreover, we show that the
acetoxylated α-amino acids can be easily converted to γ-
mercaptoamino acids, which are useful mediators of NCL for
protein synthesis.
product for the L-valine substrates was the monoacetoxylated
form (2a and 3a) which was isolated in 75% and 72% yields for
the methyl and tert-butyl esters respectively (Figure 2). It is
noteworthy that acetoxylation occurred selectively on the pro-R
γ-C of Val. For isoleucine, acetoxylation occurred exclusively on
the primary γ-C (4a and 5a). Acetoxylation of L-threonine
substrates also yielded the desired products 6a and 7a in
excellent yields. The tert-butyl and acetate groups from
compounds 6a and 7a could be removed using orthogonal
methods. This would provide amino acid derived, chemically
differentiated 1,2-diols which have great synthetic value for the
synthesis of complex natural products.
The γ-acetoxylated amino acids are precursors to γ-mercapto
amino acid derivatives. Using conventional synthetic proce-
dures as shown in Scheme 1, the γ-acetoxy-MICA-Val-O-tBu
Scheme 1. Conversion of γ-AcO-valine to γ-Mercaptovaline
Derivative by Conventional Procedures
Built on the initial success of our MICA directing group for
C−C bond formation,15 we prepared MICA-protected Val,
Thr, and Ile esters for use as substrates for direct Pd-catalyzed
γ-C acetoxylation. Indeed, when these MICA-amino acid esters
were treated with the oxidizing agent PhI(OAc)2 (2.5 equiv) in
toluene (0.25 M) in the presence of the catalyst Pd(OAc)2 (0.1
equiv) and AcOH (1 equiv) at 90 °C for 24−36 h, the
acetoxylation products were obtained in very good yields
(Figure 2). Similar to C−C bond formation,15 the predominant
ester was first converted to its Boc-protected form 9, with
recovery of the directing group, 5-methylisoxazole-3-carbox-
ylate, as the methyl ester (9a) in two steps. After removal of the
O-acetyl group, the exposed hydroxyl was mesylated, followed
by treatment with thioacetate to furnish the nucleophilic
substitution product 10. Simple deprotection and reprotection
procedures would then lead to Nα-Boc-Val(γ-SSCH3)-OH 11,
which can be coupled in solid-phase peptide synthesis as the N-
ter residue of a peptide for NCL.
Previously, Baba et al. developed a procedure to transform
alkyl acetates to thioethers using indium triiodide catalyst.16 We
found that, using this procedure, the acetoxy group in the C−H
functionalization products could also be directly converted to
benzylthioether. As shown in Scheme 2, treatment of MICA-
Ile(γ-OAc)-OMe 4a with benzylthiotrimethylsilane and indium
Scheme 2. Conversion of γ-AcO-isoleucine to the γ-
Mercaptoisoleucine Derivative Using a New Procedure
Figure 2. MICA-directed acetoxylation of Val, Ile, and Thr. All of the
reactions were carried out in a Teflon-sealed tube (see the Supporting
Information for experimental details).
B
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ο
triiodide in dry toluene at 90 C for 5 h gave directly MICA-
Ile(γ-SBn)-OMe 12 in 91% isolated yield. In a similar way, the
acetoxylated Val product 2a was also successfully converted to
the benzylthioether form 16. However, this procedure did not
work for the acetoxylated Thr derivative 6a, possibly due to the
liability of the tert-butyl ether to the Lewis acid catalyst at
elevated temperatures. Nevertheless, we can use the conven-
tional method as shown in Scheme 1 for the preparation of the
γ-mercaptothreonine derivative (Scheme S1).
synthesis of compound 18 is shown in the Figures S1 and S2.
The wild-type Xenopus laevis histone H3(1−111) MES
thioester 17 was prepared through the use of the well-
established intein splicing protocol. The purified protein
thioester was analyzed by C4 analytical RP-HPLC and
confirmed by ESI-MS analysis (Figures S4 and S5).
To synthesize Xenopus histone H3(C110A/K115Ac), the H3
C-terminal peptide 18 was reacted with H3(1−111)-MES 17
(Scheme 3). One milligram of H3(1-111)-MES thioester 17
and 1 mg of H3 C-terminal peptide 18 were dissolved in 1 mL
of ligation buffer containing 6 M Gdn·HCl, 0.2 M phosphate,
25 mM TCEP, and 50 mM MPAA, pH 7.6. The ligation was
done at room temperature for 9 h, and the reaction was
monitored with C4 analytical HPLC (Figure S6). The ligation
product was isolated by C4 semiprep HPLC and confirmed by
ESI-MS. A total of 0.5 mg of ligation product was obtained
(Figures S7 and S8).
Free-radical desulfurization was then performed to convert γ-
mercaptoisoleucine112 at the ligation junction to isoleucine
and also the native Cys110 to Ala. Initially, 0.5 mg of full-length
H3 was dissolved in 300 μL of buffer containing 6 M Gdn·HCl,
0.2 M phosphate. Subsequently, 100 μL of 1 M TCEP, 5 μL of
100 mM reduced glutathione, and 20 μL of 0.4 M VA-044 were
added sequentially. The final pH of the mixture was adjusted to
6.0. After 5 h reaction at 37 °C, another 20 μL of 0.4 M VA-044
was added and the reaction continued for another 3 h at 37 οC.
The final desulfurization product was obtained after C4
semiprep HPLC purification and confirmed by ESI-MS. A
total of 0.3 mg of final product H3(C110A/K115Ac) 20 was
obtained (Figure 3).
We chose to proceed with MICA-Ile(γ-SBn)-OMe 12 to
prepare the desired γ-mercapto-functionalized Ile as it has not
been synthesized previously for NCL. After MICA was changed
to Boc and the methyl ester was hydrolyzed, the benzyl
thioether was deprotected with sodium in liquid ammonia to
afford the free thiol compound 15. The S-trityl group was then
introduced to give Boc-Ile(γ-STrt)-OH 1, which can then be
coupled as the last amino acid in SPPS to give the γ-
mercaptoisoleucyl peptide for NCL.
We then continued to demonstrate the native chemical
ligation−desulfurization strategy for ligation at isoleucine in the
semisynthesis of a histone protein, Xenopus H3 (Scheme 3). As
Scheme 3. Semisynthesis of Xenopus Histone H3(C110A/
K115Ac)
for all histone proteins, H3 undergoes various post-translational
modifications (PTMs) that regulate the dynamics and
complexity of chromatin activity. Chemical synthesis of
homogeneous histone H3 and its analogues should assist the
ongoing efforts in elucidating and understanding the functional
roles of these PTMs. Previously, our group has synthesized an
H3 protein by using the thioacid capture ligation strategy.17 In
the current strategy, we designed the preparation for histone
H3 20 through the ligation between histone H3(1−111) 17,
which bears the MES thioester, and peptide H3(113−135) 18,
which has an N-terminal γ-mercapto-Ile.
Figure 3. (A) C4 analytical HPLC of the final desulfurized product
H3(C110A/K115Ac). Gradient: 0−80% buffer B in buffer A for 40
min. (B) Raw and deconvoluted ESI-MS of purified H3-
(C110AK115Ac). Average mass: calcd 15280.7, obsd 15283.
The H3 C-terminal peptide H3(112−135) 18 with sequence
H-I*HAK(Ac)RVTIMPKDIQLARRIRGERA-OH (I* de-
notes γ-HS-Ile) was synthesized manually by employing the
standard fluorenylmethyloxycarbonyl (Fmoc) SPPS chemistry
except for the last N-terminal amino acid which was added
using Boc-Ile(γ-STrt)-OH. This synthetic peptide contains a
Lys(Ac) at position 115. HPLC and ESI-MS analysis for the
In summary, we have shown that, in addition to the
previously demonstrated C−C bond formation,15 5-methyl-
isoxazole-3-carboxamide (MICA) can also be used to direct Pd-
catalyzed acetoxylation of unactivated γ C(sp3)−H bonds in
several amino acid substrates (Val, Thr, and Ile). An excellent
recent review on isoxazoles has documented metal-catalyzed
reactions of isoxazoles.18 Our work provides another new use of
C
DOI: 10.1021/acs.orglett.6b01160
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.6b01160.
Experimental procedures and detailed characterization
data of all new compounds (PDF)
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: cfliu@ntu.edu.sg.
Notes
(9) (a) Dawson, P. E. Isr. J. Chem. 2011, 51, 862. (b) Wong, C. T.;
Tung, C. L.; Li, X. Mol. BioSyst. 2013, 9, 826.
The authors declare no competing financial interest.
(10) Desai, L. V.; Hull, K. L.; Sanford, M. S. J. Am. Chem. Soc. 2004,
126, 9542.
ACKNOWLEDGMENTS
■
(11) Giri, R.; Liang, J.; Lei, J. G.; Li, J. J.; Wang, D. H.; Chen, X.;
Naggar, I. C.; Guo, C.; Foxman, B. M.; Yu, J. Q. Angew. Chem., Int. Ed.
2005, 44, 7420.
This work is supported by a GAP grant from ETPL of A*star
(ETPL-QP-19-06 to CFL).
(12) Zaitsev, V. G.; Shabashov, D.; Daugulis, O. J. Am. Chem. Soc.
2005, 127, 13154.
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