Synthesis of glyoxylyl peptides using a phosphine labile α,α′-diaminoacetic acid derivative

Article abstract of DOI:10.1016/j.tetlet.2004.07.038

We describe in this letter the preparation of a novel protected α,α′-diaminoacetic acid derivative that acts as a masked glyoxylic acid equivalent. The reagent could easily be introduced on a peptide chain using standard Fmoc/tert-butyl solid-phase methods and resisted to the TFA treatment allowing the deprotection and cleavage of the peptide. Unmasking of the glyoxylyl group was performed in solution in the presence of a phosphine.

Full text of DOI:10.1016/j.tetlet.2004.07.038

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 7163–7165
Synthesis of glyoxylyl peptides using a phosphine labile
a,a0-diaminoacetic acid derivative
Samia Far and Oleg Melnyk*
´
UMR 8525 CNRS-Universitede Lille 2, Institut de Biologie de Lille, 1 rue du Pr Calmette, 59021 Lille, France
Received 7 June 2004; revised 8 July 2004; accepted 9 July 2004
Available online 13 August 2004
Abstract—We describe in this letter the preparation of a novel protected a,a0-diaminoacetic acid derivative that acts as a masked
glyoxylic acid equivalent. The reagent could easily be introduced on a peptide chain using standard Fmoc/tert-butyl solid-phase
methods and resisted to the TFAtreatment allowing the deprotection and cleavage of the peptide. Unmasking of the glyoxylyl
group was performed in solution in the presence of a phosphine.
ꢀ 2004 Elsevier Ltd. All rights reserved.
The use of a-oxo aldehyde functionalized peptides for
the convergent synthesis of large molecular constructs
or hybrid materials has been the subject of numerous re-
ports over the past decade.1 Despite the interest of this
modification, there is a lack of general methods allowing
the synthesis of glyoxylyl peptides using mild and non-
oxidizing experimental conditions. Recently, a Fmoc-
protected a,a0-diaminoacetic acid derivative 1 (Scheme
1) acting as a masked glyoxylic acid equivalent was pre-
pared in one step from glyoxylic acid. Compound 1
could be introduced on a peptide chain after Fmoc/
tert-butyl solid-phase peptide synthesis. Deprotection
and cleavage of the peptide from the solid support using
trifluoroacetic acid were followed by unmasking of the
glyoxylyl group in the presence of a base such as DBU.2
could easily be introduced into a peptide using standard
Fmoc/tert-butyl protocols. Interestingly, unmasking
occurred under mild reducing conditions in aqueous
solution.
Glyoxylic acid derivative 2 was prepared as depicted in
Scheme 2. S-tert-Butylthio group was first introduced
on 2-mercaptoethanol using di-tert-butyl 1-(tert-butyl-
thio)-1,2-hydrazine dicarboxylate.3 Disulfide 3 was then
converted to carbamate 5 by reaction with p-nitrophenyl
chloroformate to give carbonate 4, followed by ammo-
nia treatment. Reaction of glyoxylic acid hydrate with
2equiv of carbamate 5 in refluxing toluene and in the
presence of a catalytic amount of p-toluenesulfonic acid
afforded bis[2-(tert-butyldisulfanyl)ethoxycarbonylami-
no] acetic acid 2 with a 54% yield following silica gel
chromatography.
Here we present the preparation and use of a,a0-diami-
noacetic acid derivative 2 (Scheme 1), which as for 1
Derivative 2 was then coupled to the N-terminus of pep-
tididyl resin 6, which was prepared using standard
Fmoc/tert-butyl protocols4 (Scheme 3). Deprotection
and cleavage of the peptide were performed in concen-
trated trifluoroacetic acid, which left the a,a0-diamino-
acetyl moiety unaffected. Peptide 7 was isolated in 40%
yield after RP-HPLC purification.
O
O
S
S
O
O
S
S
H
N
HN
Fmoc
OH
OH
NH
O
NH
Fmoc
O
1
2
Deprotection of the bis[2-(tert-butyldisulfanyl)ethoxy
carbonylamino]acetyl moiety is based on the reductive
cleavage of disulfide bonds. Generation of the free
thiols was expected to induce intramolecular thiirane
formation and then decarboxylation. Once generated,
the a,a0-diaminoacetyl group is known to hydrolyze
Scheme 1. a,a0-Diaminoacetic acid derivatives 1 and 2.
Keywords: a-Oxo aldehyde; Peptide; Phosphine.
*
Corresponding author. Tel.: +33 0320871214; fax: +33
0320871235; e-mail: oleg.melnyk@ibl.fr
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.07.038

7164
S. Far, O. Melnyk / Tetrahedron Letters 45 (2004) 7163–7165
ii
i
S
OH
S
O
O
NO2
OH
S
S
HS
O
3 (80%)
4 (87%)
O
H
N
S
S
S
S
O
O
O
iii
iv
S
O
O
NH2
S
NH
OH
O
5 (92%)
2 (54%)
Scheme 2. Synthesis of glyoxylic acid derivative 2. Reagents and conditions: (i) di-tert-butyl 1-(tert-butylthio)-1,2-hydrazine dicarboxylate, Et3N,
DMF, rt, overnight; (ii) 4-nitrophenyl chloroformate, Et3N, THF, 0ꢁC!rt, overnight; (iii) NH4OH 32%, CH3CN, rt, 1h; (iv) glyoxylic acid
monohydrate (0.5equiv), PTSA, toluene, reflux (Dean–Stark trap), 2h.
Boc tBu
Trt
phine hydrochloride (TCEP), which is known to be gen-
erally more efficient than DTT for the reduction of
disulfide bonds,5 permitted the clean conversion of pep-
tide 7 (22.49min) into glyoxylyl peptide 10 (11.74min).6
The product formed in this reaction after 21h at rt was
found to be identical by RP-HPLC to a reference gly-
oxylyl peptide obtained by periodic oxidation of the cor-
responding seryl precursor7 and displayed the expected
analytical characteristics. The monitoring of the reac-
tion revealed also two intermediate peaks at 19.15 and
15.03min, which corresponded probably to derivatives
8 and 9, respectively (Scheme 4). Indeed, the same prod-
ucts could be formed, isolated and characterized by
mass spectrometry when the reaction was performed at
pH5.5, experimental conditions, which permitted the
reduction of the disulfide bonds but not the intramole-
cular nucleophilic substitution.
i
H2N
H
I
L K E P V H G V NH
NovaSyn
TGR resin
6
ii, iii
O
O
H
S
S
O
S
S
O
O
N
I
L K E P V H G V NH2
NH
7
Scheme 3. Synthesis of peptide 7 using derivative 2. Reagents and
conditions: (i) Fmoc/t-Bu solid-phase peptide synthesis using TBTU/
HOBt/DIEAactivation in DMF; (ii) 2 (1.2equiv), PyBOP/DIEA,
DMF, 1h; (iii) TFA/H2O/anisole (95/2.5/2.5 by vol).
spontaneously in aqueous medium to give the a-oxo
aldehyde function.2
In conclusion, we have prepared a novel protected a,a0-
diaminoacetic acid derivative that can be easily intro-
duced into peptide using standard Fmoc/tert-butyl
methods and deprotected in aqueous solution to give a
glyoxylyl group. The glyoxylyl group was generated in
the presence of a phosphine at pH8.3. This reagent is
thus complementary to (FmocNH)2CHCO2H derivative
Deprotection was first attempted using dithiothreitol
(DTT) as reducing agent in 0.1M Tris–HCl buffer
(pH8.3). The reaction was found to be poorly efficient
as shown by RP-HPLC monitoring. Alternately and as
shown in Figure 1, the use of tris(2-carboxyethyl)phos-
Figure 1. RP-HPLC monitoring of the conversion of peptide 7 into glyoxylyl peptide 10 in the presence of TCEP in 0.1M Tris–HCl buffer pH8.3.
C18 Delta Pak 3.9 · 300mm column, eluent A: water containing 0.05% TFAby vol, eluent B: acetonitrile/water 4/1 by vol containing 0.05% TFAby
vol, linear gradient 0–100% B in 30min, 1mL/min, detection at 215nm.

S. Far, O. Melnyk / Tetrahedron Letters 45 (2004) 7163–7165
7165
O
O
H
N
S
S
H
N
S
O
O
S
S
O
O
S
O
O
TCEP
Peptide
Peptide
NH
NH
Tris-HCl buffer
(0.1 M, pH 8.3)
HS
O
O
7
8
O
H
N
HS
HS
O
O
H
O
O
Peptide
Peptide
O
NH
O
10
9
Scheme 4. Unmasking of peptide 7 in the presence of TCEP in 0.1M Tris–HCl buffer pH8.3.
2. Far, S.; Melnyk, O. Tetrahedron Lett. 2004, 45, 1271–1273.
3. Wunsch, E.; Moroder, L.; Romani, S. Hoppe-SeylerÕs Z.
1, whose unmasking requires a base such as DBU in an
organic solvent (DMF).
¨
Physiol. Chem. 1982, 363, 1461–1464.
4. Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990,
35, 161–214.
5. Getz, E. B.; Xiao, M.; Chakrabarty, T.; Cooke, R.; Selvin,
P. R. Anal. Biochem. 1999, 273, 73–80.
6. Typical deprotection procedure: the peptide (3.0lmol) was
dissolved in 5mL of 0.1M Tris–HCl buffer (pH 8.33).
Acknowledgements
`
We gratefully acknowledge le Ministere de lÕEconomie,
des Finances et de lÕIndustrie (GenHomme, Coquepuce
´
Project), CNRS, Universitede Lille 2 and Institut Pas-
Tris(2-carboxyethyl)phosphine
hydrochloride
(86mg,
teur de Lille for financial support. We also acknowledge
Anthony Romieu for helpful discussions.
0.3mmol, 100equiv) was added and the mixture was stirred
at room temperature for 21h. Aliquots (95lL) of the
reaction mixture were mixed with 5lL of acetic acid and
injected on a C18 Delta Pak column (3.9 · 300mm,
experimental conditions specified in Fig. 1) for HPLC
monitoring.
References and notes
1. Melnyk, O.; Fehrentz, J.-A.; Martinez, J.; Gras-Masse, H.
Peptide Sci. 2000, 55, 165–186.
7. Geoghegan, K. F.; Stroh, J. G. Bioconjugate Chem. 1992, 3,
138–146.