Facile synthesis of α-hydroxy carboxylic acids from the corresponding α-amino acids

Article abstract of DOI:10.1016/j.tetlet.2014.05.090

An effective and improved procedure is developed for the synthesis of α-hydroxy carboxylic acids by treatment of the corresponding protonated α-amino acid with tert-butyl nitrite in 1,4-dioxane-water. The amino moiety must be protonated and located α to a carboxylic acid function in order to undergo initial diazotization and successive hydroxylation, since neither β-amino acids nor acid derivatives such as esters and amides undergo hydroxylations. The method is successfully applied for the synthesis of 18 proteinogenic amino acids.

Full text of DOI:10.1016/j.tetlet.2014.05.090

Accepted Manuscript
Facile synthesis of α-hydroxy carboxylic acids from the corresponding α-amino
acids
Nicolai Stuhr-Hansen, Shahrokh Padrah, Kristian Strømgaard
PII:
S0040-4039(14)00911-3
DOI:
http://dx.doi.org/10.1016/j.tetlet.2014.05.090
TETL 44681
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 April 2014
13 May 2014
22 May 2014
Please cite this article as: Stuhr-Hansen, N., Padrah, S., Strømgaard, K., Facile synthesis of α-hydroxy carboxylic
acids from the corresponding α-amino acids, Tetrahedron Letters (2014), doi: http://dx.doi.org/10.1016/j.tetlet.
2014.05.090
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Facile synthesis of α-hydroxy carboxylic acids from the corresponding α-
amino acids
Nicolai Stuhr-Hansen,*a,b Shahrokh Padrah,a Kristian Strømgaarda
aDepartment of Drug Design and Pharmacology, University of Copenhagen,
Universitetsparken 2, DK-2100 Copenhagen, Denmark.
bDepartment of Chemistry, Faculty of Sciences, University of Copenhagen,
Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
E-mail: nsh@chem.ku.dk
_________________________________________________________________
Abstract.
An effective and improved procedure is developed for the synthesis of α-hydroxy
carboxylic acids by treatment of the corresponding protonated α-amino acid with tert-butyl
nitrite in 1,4-dioxane-water. The amino moiety must be protonated and located α to a
carboxylic acid function in order to undergo initial diazotization and successive
hydroxylation, since neither β-amino acids nor acid derivatives such as esters and amides
undergo hydroxylations. The method is successfully applied for the synthesis of 18
proteinogenic amino acids.
Keywords: α-Hydroxy carboxylic acids, diazotization, hydroxylation, depsipeptides.
__________________________________________________

Peptides containing ester bonds in their backbone, which are known as depsipeptides,1
are found in Nature, often from marine sources, as cyclic depsipeptides2 with many
exciting biological properties. Most notably, romidepsin is a histone deacetylase (HDAC)
inhibitor and an approved drug for the treatment of cutaneous T-cell lymphoma.3
Depsipeptides are potential drug targets displaying effects on drug resistance in tumor
cells4 and are potent antitumor agents.5,6,7 Furthermore, they exhibit antitubercular activity8
and inhibit S1-serine protease.9 In addition, natural depsipeptides such as antillatoxin,10
aomamide A,11 apratoxins,12 lyngbyabelins,13 spiruchostatin14 and stigonemapeptin15 have
been isolated from various species.
Amide-to-ester mutations16 have been introduced in proteins as a method17 to evaluate, for
example, the effects of specific protein backbone-backbone hydrogen bonds on protein
folding. By replacing the backbone amide with an ester,18 the hydrogen bonding ability of
the amide NH is removed, and the carbonyl becomes a much weaker hydrogen bond
acceptor19 in accordance with theoretical investigations.20 Recently, we employed amide-
to-ester mutations both in peptide ligands and in proteins to evaluate the importance of
backbone hydrogen bonds for protein-protein interactions, specifically the interaction of
peptide ligands with PSD-95/Discs-large/ZO-1 (PDZ) domains.21,22
The synthesis of depsipeptides has typically been conducted by solid-phase peptide
synthesis,23 introducing the appropriate ester moieties by incorporating α-hydroxy
carboxylic acids either using peptide coupling reagents24,25 or by application of the
hexafluoroacetone bidentate protecting/activating technique.26,27 α-Hydroxy carboxylic
acids have also been used as synthetic tools for enantiomeric additions to aldehydes

facilitating the synthesis of optically active alcohols28 and as monomers for poly(α-hydroxy)
acids29 with potential for production of biodegradable polymers.30
Only a few appropriately side-chain protected α-hydroxy carboxylic acids for use in Boc-
based solid phase chemistry are commercially available in acceptable quantities and
purities, these include: glycolic acid (HO-Gly-OH), (S)-lactic acid (HO-Ala-OH), (S)-2-
hydroxyisocaproic acid (HO-Leu-OH), (2S,3R)-2-hydroxy-3-methylpentanoic acid (HO-Ile-
OH), (S)-2-hydroxy-3-methylbutyric acid (HO-Val-OH, valic acid), (S)-3-phenyllactic acid
(HO-Phe-OH), and (S)-3-(benzyloxy)-2-hydroxypropanoic acid (HO-Ser(Bn)-OH).
Furthermore, the synthetic challenges associated with obtaining side-chain protected α-
hydroxy carboxylic acids for incorporation in depsipeptides have led researchers to mutate
peptides and proteins, i.e., not using the native residues, due to the unavailability of these
building blocks. Even though side-chain protected α-hydroxy carboxylic acids are versatile
building blocks with a broad range of applications, they have previously been synthesized
exclusively by a diazotization procedure using HNO2 generated from sodium nitrite under
protic conditions.31 In some cases these conditions are too harsh for side-chain protecting
groups,32 and in our hands only a limited selection of α-hydroxy carboxylic acids could be
isolated in satisfactory yields and purities using this diazotization procedure. It was
therefore desirable to employ milder conditions for the synthesis of these important
synthons and herein we describe the synthesis of α-hydroxy carboxylic acids directly from
appropriately side-chain protected Boc-solid-phase peptide chemistry compatible α-amino
acids (Scheme 1):

In order to develop a general method for the synthesis of α-hydroxy carboxylic acids from
the possible 19 proteinogenic amino acids which have primary amino groups, we exploited
the mildest possible diazotization conditions. Since these hydroxy acids are to be applied
primarily for construction of depsipeptides or proteins involving Boc-based solid-phase
peptide synthesis,33 it was therefore necessary to utilize side-chain protecting groups that
tolerate TFA, but on the other hand, that can be cleaved with HF34 in the final step. The
standard properly side-chain protected Boc-amino acids used in Boc-based SPPS fulfilled
these requirements. On treating these compounds with TFA the protonated amine salts
were obtained. Because diazotization of these amino acids with aqueous sodium nitrite in
the presence of a strong acid had not been applied for the synthesis of all nineteen α-
hydroxy carboxylic acids,32 milder alternative conditions were sought in order to avoid the
formation of undesired decomposition products. Treatment of the TFA-salts of the
proteinogenic amino acids with one equivalent of tert-butyl nitrite in a 1:1 mixture of water-
1,4-dioxane (Scheme 1) gave 18 of the corresponding α-hydroxy carboxylic acids (Table
1).35 All the α-hydroxy carboxylic acids were isolated directly by normal phase
chromatography, except HO-Arg(Tos)-OH and HO-His(Bom)-OH, which were purified by
HPLC. Identical optical rotations of synthesized vs. commercially available HO-Ala-OH
(approximately +2.5; c=2.5 in water, 20 °C) and HO-Phe-OH (approximately +21; c=2.0 in
water, 20 °C) were recorded. By performing chiral HPLC on a Crownpack CR(-) column
(Chiral Technologies Europe) to examine the optical purities of synthesized HO-Ala-OH,
HO-Leu-OH, HO-Val-OH, and HO-Phe-OH vs. the corresponding four commercially
available hydroxy acids, no indication of inversion of the configurations were observed.
The use of synthesized R-HO-Phe-OH and R-HO-Val-OH as internal standards resulted in

different retention times being demonstrated compared with the corresponding S-hydroxy
acids, with clear base-line separations. Based on these chirality measurements it can be
concluded that the enantiomeric excesses of the examined synthetic α-hydroxy carboxylic
acids were above 99% with full retention of configuration in agreement with reported
literature values.36 These observations allowed us to postulate a mechanism proceeding
via intramolecular hydroxy group attack at the diazonium ion generated by diazotization of
the amine, followed by solvolysis of the intermediate oxiranium ion (Scheme 2).
Table 1
i. TFA, rt, 15 min
Boc-AA(Pg)-OH
HO-AA(Pg)-OH
t
ii. -BuONO,
1,4-dioxane-H 2O (1:1)
rt, 1 h
HO-AA(Pg)-OH
HO-Ala-OH
Yield
58%
Yield
49%
HO-AA(Pg)-OH
HO-Lys(2-Cl-Z)-OH
HO-Asp(cHx)-OH
HO-Glu(cHx)-OH
35%
62%
HO-Ile-OH
21%
53%
15%
41%
36%
HO-Phe-OH
HO-Met-OH
HO-Asn-OH
51%
-
HO-Ser(Bn)-OH
HO-Thr(Bn)-OH
HO-Gln-OH
HO-His(Bom)-OH
72%
HO-Tyr(Bn)-OH
HO-Arg(Tos)-OH
HO-Trp(CHO)-OH
27%
8%
15%
18%
HO-Cys(4-Me-Bn)-OH
Pg = Side-chain protecting group
Boc-glutamine (1) could not be converted into the corresponding α-hydroxy acid (HO-Gln-
OH). Almost no reaction was observed utilizing the general conditions. However, upon

concentration and prolonged stirring, glutamic acid (2) (61% yield) and an inseparable
approximately 4:1 mixture of 5-oxo-proline (3) and 5-oxo-tetrahydrofuran-2-carboxylic acid
(4) (22% overall yield, Scheme 3) was isolated by HPLC, but HO-Gln-OH was not formed.
Compound 2 is presumably formed by initial diazotization of the amide function followed by
hydrolysis.37 Diazotization of either of the two NH2 moieties and spontaneous ring closure
affords 3 independent of which nitrogen was diazotized. Compound 4 may be formed by
lactonization of HO-Glu-OH formed by diazotization and successive hydroxylation of both
NH2 moieties (see Supporting Information).
In order to examine the importance of a free carboxylic acid in the α-amino acid for the
diazotization/hydroxylation procedure, we subjected the ethyl ester of alanine to the
diazotization conditions. No hydroxylated products were observed and the unreacted ester
could be isolated in quantitative yield. This shows that a free carboxylic acid moiety is
essential for formation of the diazonium ion and therefore for overall hydroxylation to
occur. The importance of the position of the free carboxylic acid moiety for diazotization to
occur was demonstrated by attempting the reaction with β-phenylalanine, which, in
contrast to α-phenylalanine, did not undergo reaction and unreacted starting material was
isolated in quantitative yield. The latter experiments demonstrate that an α-carboxylic acid
assisted formation of the diazonium ion was key in the formation of the corresponding
hydroxylated compounds. Furthermore, when attempting reactions of zwitterionic amino

acids in the absence of additional acid, diazotization was not observed. This suggests that
for diazotization to occur and subsequent hydroxylation, the amine had to be protonated
and needed a carboxylic acid function present on the α-carbon.
One of the advances utilizing the presented diazotization procedure is the mild conditions
employed, since it proved possible to prepare the α-hydroxy carboxylic acids: HO-Met-OH,
HO-Cys(4-Me-Bn)-OH and HO-His(Bom)-OH by direct diazotization using these
conditions, which is not possible utilizing sodium nitrite based procedures from Boc-
compatible amino acids. In these cases it was necessary32 to prepare the hydroxyl acids
using ‘non-diazo conditions’ via two-step routes due to by-product formation. Direct HPLC
purification was possible for all the α-hydroxy carboxylic acids, but traces of nitrites and
1,4-dioxane proved to shorten the life-time of reverse phase columns. Therefore, normal
phase purification is recommended in general cases.
In conclusion, we have synthesized 18 α-hydroxy carboxylic acids from the corresponding
proteinogenic α-amino acids using mild diazotization conditions. All the products were
synthesized directly from the appropriately protected Boc-chemistry compatible α-amino
acids. The only anomaly apart from the logical exception proline was glutamine, which
afforded glutamic acid, 5-oxo-proline, and 5-oxo-tetrahydrofuran-2-carboxylic acid instead
of the corresponding α-hydroxy carboxylic acid (HO-Glu-OH). Furthermore, a short
mechanistic survey revealed that diazotization and successive hydroxylation of an amine
using tert-butyl nitrite only took place when a free carboxylic acid was present at the α-
carbon.

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