Mild oxidative cleavage of 9-BBN-protected amino acid derivatives

Article abstract of DOI:10.1002/ejoc.201500361

Protection of the amino acid moiety using 9-BBN is an effective method to enable side chain manipulations in synthesis of complex amino acids. We investigated the standard, mild method for deprotection of the 9-BBN group in methanolic chloroform, and found that it relies on a slow oxidation mediated by molecular oxygen. Building on this insight, we have developed a method that allows for a fast and selective deprotection using simple peroxy acid reagents. After Fmoc protection, products were isolated in >90% yield for a series of amino acid derivatives, including a galactosylated derivative of hydroxylysine. A representative set of 9-BBN-protected amino acid derivatives were efficiently deprotected using peracid reagents in excellent yields. Deprotection is orthogonal with several common protecting groups. Its tolerance of highly acid sensitive groups, such as trityl-protected amides and glycosidic linkages, is especially notable.

Full text of DOI:10.1002/ejoc.201500361

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DOI: 10.1002/ejoc.201500361
Mild Oxidative Cleavage of 9-BBN-Protected Amino Acid Derivatives
Tobias Ankner,[a] Thomas Norberg,[a] and Jan Kihlberg*[a]
Keywords: Amino acids / Protecting groups / Oxidation
Protection of the amino acid moiety using 9-BBN is an effec- developed a method that allows for a fast and selective de-
tive method to enable side chain manipulations in synthesis
of complex amino acids. We investigated the standard, mild
method for deprotection of the 9-BBN group in methanolic
chloroform, and found that it relies on a slow oxidation medi-
ated by molecular oxygen. Building on this insight, we have
protection using simple peroxy acid reagents. After Fmoc
protection, products were isolated in Ͼ90% yield for a series
of amino acid derivatives, including a galactosylated deriva-
tive of hydroxylysine.
tions and glycosylation. However, use of cupric chelates in-
volved practical difficulties such as tedious workup and iso-
lation of products, and also steps that required carefully
controlled conditions so as not to yield side-products. We
therefore developed a route to galactosylated hydroxylysine
based on use of the 9-BBN group for protection of the α-
amino acid moiety.[5b] Unfortunately, when this procedure
was used on a multigram scale the yield for cleavage of the
9-BBN group in a chloroform/methanol mixture dropped
from just over 90% obtained on a small scale[5b] to a mere
38%. We therefore set out to investigate the mechanism be-
hind the cleavage of the 9-BBN group in chloroform and
methanol. Herein we report our findings on the require-
ments for deprotection using chloroform-methanol, as well
as an efficient and selective procedure for oxidative cleavage
that consistently provides high yields regardless of scale.
Introduction
Synthesis of organic compounds frequently requires the
use of protecting groups to achieve selectivity between posi-
tions of similar reactivity.[1] In the construction of complex
amino acid derivatives, protection of the amino acid moiety
via metal or metaloid chelation is an effective way to enable
selective side-chain manipulations. In particular, the use of
9-borabicyclononane (9-BBN) has proven successful as the
formed complex displays surprising stability towards
various reaction conditions and is also soluble in organic
solvents.[2] The 9-BBN group has traditionally been re-
moved under harsh conditions by exchange with ethylenedi-
amine[3] or by hydrolysis with aqueous HCl,[4] making
either of these two methods unsuitable for amino acid de-
rivatives containing additional, sensitive functionalities.
However, a decade ago it was reported that the 9-BBN
group is slowly cleaved by stirring in a mixture of methanol
and chloroform,[5] and this has since become a standard
protocol for deprotection.[6] The exact mechanism behind
this reaction is not understood, but it has been speculated
that residual HCl in the chloroform accelerates the depro-
tection or that precipitation drives the reaction.[5a]
Results
Modification of the reaction conditions for cleavage of
the 9-BBN group in methanolic chloroform by addition of
potassium carbonate, or various acids,[10] did not result in
any appreciable increase in the rate of deprotection, nor in
yield. This led us to believe that a mechanism completely
different from the ones proposed was operating and we
suspected that oxidation was responsible for the erratic
behavior. Interestingly, careful distillation of the solvents
under argon, and running the reaction in darkness under
argon, resulted in complete inhibition of the deprotection
of the 9-BBN complex of phenylalanine in chloroform/
methanol (Figure 1). Addition of 2,6-di-tert-butyl-4-meth-
ylphenol (BHT, 3 equiv.) as an inhibitor of radical reactions
when deprotection was carried out under air resulted in a
considerably slower reaction than in the absence of BHT,
suggesting that either oxygen or radicals were involved in
the reaction. However, no difference in the rate of deprotec-
tion was observed upon addition of tert-butyl peroxy-
We have a vested interest in the synthesis of glycosylated
derivatives of hydroxylysine as part of our efforts to under-
stand the role played by glycopeptide fragments from
type II collagen in development of rheumatoid arthritis.[7]
Procedures for synthesis of β-d-galactosylated derivatives of
(5R)-5-hydroxy-l-lysine, reported initially from our[8] and
other laboratories[9] relied on formation of a cupric chelate
of hydroxylysine followed by protective group manipula-
[a] Department of Chemistry – BMC, Uppsala University,
Box 576, 75123 Uppsala, Sweden
E-mail: Jan.Kihlberg@kemi.uu.se
http://kemi-wp2.beta.uu.se/research/physical-organic-
chemistry/groups/kihlberg-group/
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201500361.
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T. Ankner, T. Norberg, J. Kihlberg
FULL PAPER
benzoate as a radical initiator, indicating that cleavage was Table 1. Attempted oxidation of the 9-BBN complex of phenyl-
alanine (1a) with different oxidative reagents.
not mediated by radicals. In an effort to increase the rate
of the deprotection, the reaction was stirred under O2; this
did however not increase the rate appreciably and was not
pursued as a preparative procedure. These initial studies
lend support to our hypothesis that oxygen is involved in
the deprotection and may explain why we, and others, have
experienced irreproducible results. The decreased yield
could be explained by assuming that the lower surface-to-
volume ratio on larger scale effectively decreases the
available oxygen, thus inhibiting the reaction. It should,
however, be mentioned that reports in the literature on the
reactivity of the 9-BBN group to oxidants are conflicting.
While some authors claim that it is resistant towards m-
chloroperoxybenzoic acid (MCPBA)[2a] and oxygen,[6d]
others note its sensitive nature under such conditions.[2b]
Entry Reagent[a]
Result
1
2
3
4
5
6
7
8
CAN
no reaction
no reaction
slow conversion
no reaction
SelectFluor®
TBHP
UHP
UHP and TFA
PAA, 3 equiv.
MCPBA, 3 equiv.
TFPAA, 3 equiv.
no reaction
100% conversion in 3 h
100% conversion in Ͻ 1 h
100% conversion in Ͻ 10 min
[a] See supporting information for details.
Figure 1. Consumption of 9-BBN-protected phenylalanine (1a) un-
der different conditions. The indicated reagents/solvent were added
to a solution of 1a in purified and degassed CHCl3. The concentra-
tion of 1a relative to time = 0 min was determined using LCMS by
comparing with an internal standard (cf. Supporting Information).
Figure 2. Consumption of 9-BBN-protected phenylalanine (1a) in
presence of different peracids. Stirring of 1a in a CHCl3/MeOH
mixture has been included for comparison. The concentration of
1a relative to time = 0 min was determined using LCMS by com-
paring with an internal standard (cf. Supporting Information).
Inspired by the above findings, we exposed the 9-BBN
derivative of phenylalanine (1a) to a set of different oxidat-
ive reagents (Table 1). While ceric ammonium nitrate
(CAN) and 1-(chloromethyl)-4-fluoro-1,4-diazoniabicyclo-
[2.2.2]octane ditetrafluoroborate (SelectFluor®)[11] did not
Some deglycosylation was detected when galactosylated
cleave the 9-BBN group (entries 1 and 2), the use of tert- hydroxylysine (5a, Table 2) was deprotected with TFPAA
butyl hydrogen peroxide (TBHP, entry 3) led to a slow and this was suspected to result from the acidic conditions.
cleavage, albeit too slow for practical use. The urea– Addition of sodium hydrogenphosphate (Na2HPO4) com-
hydrogen peroxide complex (UHP), either alone or in com- pletely prevented this undesirable side reaction. To demon-
bination with trifluoroacetic acid (TFA), showed no reactiv- strate the generality of this method on a preparative scale
ity (entries 4 and 5). Peracetic acid (PAA), on the other (Ն 1 mmol), 9-BBN-protected amino acid derivatives 1a–5a
hand, gave a relatively fast and clean deprotection at room were dissolved in dichloromethane or acetonitrile and then
temperature. Peroxy acid based reagents were therefore pur- treated with a solution TFPAA in the presence of Na2HPO4
sued further, which revealed that both MCPBA and trifluo- for 1 h at room temperature (Table 2).[12] Using this pro-
roperacetic acid (TFPAA) gave an even faster cleavage of cedure, phenylalanine derivative 1a was deprotected within
1a than PAA. To further investigate the deprotection of 1a minutes and Fmoc protection gave 1b in 92% isolated yield
using peracids, we followed the reactions over time and over two steps after column chromatography. Gratifyingly,
compared PAA, MCPBA and TFPAA (Figure 2). TFPAA the acid-labile 9-BBN derivatives of tert-butyl glutamic acid
mediated cleavage showed a considerably shorter half-life (2a) and N-Boc lysine (3a) were both deprotected cleanly
(appr. 2 min) than both MCPBA (20 min) and PAA and then Fmoc reprotected to give 2b and 3b in 92% and
(80 min). In contrast, deprotection using the original condi- 93% yields, respectively (entries 2 and 3). Even the trityl
tions, i.e. by stirring in methanolic chloroform under air, group, which was selected because of its very high sensiti-
showed a substantially longer half-life (10 h).
vity towards acid, allowed successful conversion of pro-
2
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Mild Oxidative Cleavage of 9-BBN-Protected Amino Acids
General Procedure for Deprotection: The 9-BBN-protected amino
acid (1 mmol) was vigorously stirred in acetonitrile (12 mL) at
room temperature. Na2HPO4 (3 mmol, 426 mg) was added to this
solution followed by the dropwise addition of trifluoroperacetic
acid (6 mL, 0.5 m in acetonitrile).[13] The reaction mixture was
stirred until the starting material was consumed, as judged by TLC
and/or LC/MS, then quenched by the addition of a 20% solution
of Na2SO3 in water (1 mL). The solvent was removed on a rotary
evaporator, the residue was redissolved in dioxane (15 mL) and
water (5 mL), treated with solid NaHCO3 (5 mmol, 420 mg) and
cooled to 0 °C. Fmoc-OSu (1.2 mmol, 405 mg) was added in one
portion and the reaction was stirred at water-ice bath temperature
for 30 min, brought to room temperature and stirred until full con-
version of the amino acid had been obtained. The volatiles were
removed, additional water was added (20 mL) and the pH was ad-
justed to 3 with 1 m KHSO4. The water phase was extracted with
dichloromethane (4ϫ 15 mL), the pooled organic extracts were
dried with Na2SO4 and concentrated. The crude material was puri-
fied with flash chromatography using a dichloromethane/methanol/
formic acid (0.1%) gradient starting with 1% and ending with 10%
methanol.
tected glutamine 4a to 4b (entry 4). This further underlines
the high degree of orthogonality of our procedure with
highly acid sensitive protective groups. Finally, 9-BBN-pro-
tected galactosylated hydroxylysine 5a (entry 5) was cleanly
converted into the corresponding Fmoc-protected amino
acid 5b in 94% yield on a 1.5 mmol scale. It should be em-
phasized that this constitutes a vast improvement over our
earlier large-scale attempts that gave 5b in a mediocre 38%
yield.
Table 2. Isolated yields obtained in oxidative cleavage of 9-BBN
derivatives 1a–5a, followed by protection to give the corresponding
Fmoc-amino acids 1b–5b.
Fmoc-protected amino acids 1b–5b obtained as products showed
spectroscopic data in accordance with published data. Copies of 1H
and 13C NMR spectroscopic data are provided in the Supporting
Information.
Acknowledgments
This project is financially supported by the Swedish Foundation
for Strategic Research. The authors thank AstraZeneca R&D,
Mölndal, Sweden for contributing equipment that allowed this pro-
ject to be performed successfully.
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[a] All reactions were performed on a 1 mmol scale unless otherwise
[b]
[c]
stated.
Performed on 8 mmol scale (2.96 g).
Performed on
1.5 mmol scale (1.12 g).
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Conclusions
We have developed a fast and reliable method to selec-
tively deprotect 9-BBN-protected amino acid derivatives by
oxidation with trifluoroperoxy acetic acid (TFPAA). De-
protection is performed under very mild conditions, is com-
patible with sensitive structural motifs such as glycosidic
linkages, and shows orthogonality with acid labile protect-
ing groups such as N-Boc, O-tert butyl esters and even tri-
tyl-protected amides. This work thus provides the organic
chemistry community with a method that significantly ex-
pands the scope for use of the versatile 9-BBN protecting
group. The mechanism of the oxidative deprotection is un-
der further investigation.
Experimental Section
General: Details regarding synthesis of the 9-BBN-protected amino
acids 1a–5a and complete characterization of these compounds are
provided in the supporting information.
[6] a) A. Y. Shaikh, S. Das, D. Pati, V. Dhaware, S. Sen Gupta, S.
Hotha, Biomacromolecules 2014, 15, 3679–3686; b) T. Koop-
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[10]
[11]
Water, TFA, acetic acid, pyridine, BF3·OEt2, ammonium for-
mate were added without observing any increase in deprotec-
tion rate.
SelectFluor is known to be a potent oxidant and a source of
electrophilic fluorine.
®
[12] Over oxidation of reactive amino acids is a concern when using
strong oxidizing agents such as TFPAA. Not surprisingly, 9-
BBN-protected tryptophan yielded only a cyclized, oxidized
product upon exposure to TFPAA. We assume that substrates
containing sulfur, e.g. methionine, will also be oxidized.
[13] TFPAA was generated immediately before use by adding
TFAA (5 mmol, 695 μL) to a stirred suspension of urea-
hydrogen peroxide (5 mmol, 470 mg) in acetonitrile (8 mL). Af-
ter 20 min the volume of the TFPAA solution was adjusted to
10 mL.
Received: March 18, 2015
Published Online: ᭿
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Mild Oxidative Cleavage of 9-BBN-Protected Amino Acids
Amino Acid Protecting Groups
T. Ankner, T. Norberg,
J. Kihlberg* ...................................... 1–5
Mild Oxidative Cleavage of 9-BBN-Pro-
tected Amino Acid Derivatives
A representative set of 9-BBN-protected
amino acid derivatives were efficiently de-
protected using peracid reagents in excel-
lent yields. Deprotection is orthogonal with
several common protecting groups. Its
tolerance of highly acid sensitive groups,
such as trityl-protected amides and glycos-
idic linkages, is especially notable.
Keywords: Amino acids
groups / Oxidation
/
Protecting
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