Diazo ester insertion in NH bonds of amino acid derivatives and insulin catalyzed by water-soluble iron and ruthenium porphyrin complexes (FeTSPPCl) as application of carbenoid transfer in aqueous media

Article abstract of DOI:10.1016/j.molcata.2015.06.028

The metal complex FeTSPPCl (5,10,15,20-tetrakis)-(4-sulfonato-phenyl)-porphyrin-iron(III) chloride is an active catalyst for carbenoid insertion in NH bonds of aminoacid derivatives in aqueous media. A variety of diazoacetates and methyl diazophosphonate

Full text of DOI:10.1016/j.molcata.2015.06.028

Journal of Molecular Catalysis A: Chemical 407 (2015) 194–203
Contents lists available at ScienceDirect
Journal of Molecular Catalysis A: Chemical
journal homepage: www.elsevier.com/locate/molcata
Diazo ester insertion in N H bonds of amino acid derivatives and
insulin catalyzed by water-soluble iron and ruthenium porphyrin
complexes (FeTSPPCl) as application of carbenoid transfer in aqueous
media
a
a
a
a
b
Hassan F. Srour , Paul Le Maux , Soizic Chevance , Daniel Carrié , Nicolas Le Yondre ,
a,∗
Gérard Simonneaux
UMR 6226 Institut Sciences Chimiques de Rennes, Campus de Beaulieu, Université de Rennes 1, 35042 Rennes, France
a
b
CRMPO, Campus de Beaulieu, Université de Rennes 1, 35042 Rennes, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 April 2015
Received in revised form 4 June 2015
Accepted 23 June 2015
Available online 8 July 2015
The metal complex FeTSPPCl (5,10,15,20-tetrakis)-(4-sulfonato-phenyl)-porphyrin-iron(III) chloride is
an active catalyst for carbenoid insertion in N H bonds of aminoacid derivatives in aqueous media. A
variety of diazoacetates and methyl diazophosphonate were used as carbenoid precursors. The com-
mercially available iron porphyrin complex can also selectively catalyze alkylation of the N-terminus of
insulin (chain B).
©
2015 Elsevier B.V. All rights reserved.
Keywords:
Catalytic NH insertion
Iron porphyrin
Diazo ester
Amino ester
Insulin
1
. Introduction
the carbapenem nucleus via rhodium-catalyzed insertion of a keto
carbenoid into the N H bond of the ␤-lactam [6]. More gener-
ally, this insertion reaction can lead to the synthesis of ␣-amino
esters, dipeptides, and nitrogen-containing heterocycles. The cat-
alytic preparation of optically active amines, recently reported
by Zhou et al. [7] is also a nice example of application in asym-
metric synthesis. Protein modification through alkylation of the
N-terminal ␣-amino acid is also a potentially useful approach such
as recently reported by the group of Che [8]. Among the previously
reported metal-catalyzed carbenoid transfer, selective tryptophan
modification with rhodium carbenoids [9,10] and terminal amino
acid modification of proteins with ruthenium porphyrins have been
previously explored [11], although in the latter case, a stoichiomet-
ric amount of the metalloporphyrin was necessary.
The most common bioconjugations are coupling of a small
molecule (such as biotin or a fluorescent dye) to a protein. Usual
types of bioconjugation chemistry are amine coupling of lysine
amino acid residues (typically through amine-reactive succin-
imidyl esters) and sulfhydryl coupling of cysteine residues (via a
sulfhydryl-reactive maleimide) [1]. Generally, the preparation of
bifunctional agents involves multistep syntheses, and their sub-
sequent incorporation into biomolecules is often hampered by
cross-reactivity or nonspecific interactions with other functional
groups present. Thus, the quest for novel, efficient strategies for
the synthesis of functional agents, and their incorporation into
biomolecules, has led to a burgeoning interest in “metallocar-
benoid” chemistry [2].
We reported the first success in the use of ruthenium porphyrin
The catalytic N H insertion of ␣-diazocarbonyl compounds
is a powerful organic reaction which has various useful appli-
catalysts for intermolecular insertion of diazo derivatives into N H
bonds in 1997 [12]. Since then, we [13,14] and others [11,15–18]
have found that ruthenium and iron porphyrins are active cata-
lysts, for carbenoid transfer, not only in organic solvents but also
in protic solvents and water. Although, there are examples show-
ing reactions with diazoacetonitrile [19] diazomethyl phosphonate
[20] and trifluoro diazomethane [21] in organic solvents, ethyl dia-
cations [3–5]. One of the most successful intramolecular N
H
insertions has been the conversion of a penicillin analogue into
∗ Corresponding author. Fax: +33 223235637.
E-mail address: gerard.simonneaux@univ-rennes1.fr (G. Simonneaux).
http://dx.doi.org/10.1016/j.molcata.2015.06.028
1
381-1169/© 2015 Elsevier B.V. All rights reserved.

H.F. Srour et al. / Journal of Molecular Catalysis A: Chemical 407 (2015) 194–203
195
2-diazoacetate as a pale yellow oil (897 mg, 90%, two steps). 1
H
zoacetate has been mainly used as the carbenoid precursors in
water.
In this paper, we report NH insertion of amino esters using
NMR (CDCl , 400 MHz, ppm): ␦ 4.83 (s, 1H, CH N ), 5.25 (s, 2H,
3
2
13
CH ), 7.47 (d, J = 8.02 Hz, 2H), 7.63 (d, J = 7.63 Hz, 2H). C NMR
2
various diazo esters [5] as reagents and the site selective mod-
ification of terminal NH2 group of insulin using water-soluble
(CDCl , 125 MHz, ppm): ␦ 46.48 (CH N ), 65.56 (CH ), ␦ 123.05
3
2
2
(CF ), 125.65 (CHPh), 128.23 (CHPh), 130.57 (Cq-CF ), 140.06 (Cq-
3
3
19
5
,10,15,20-tetrakis-(4-sulfonato-phenyl)-porphyrin-iron(III) chlo-
CH ), 166.57 (C O). F NMR (CDCl , 400 MHz, ppm): ␦ −62.67
2
3
+
ride (FeTSPPCl) 1 as catalyst (Scheme 1). Iron porphyrin-catalyzed
NH insertions have been previously studied in organic sol-
vents [15–18]. Aviv and Gross [18] also reported intermolecular
7
2 2 3
(s). HR-MS (m/z): calculated mass for C10H N O F Na [M + Na] :
267.03573, found m/z: 267.0356 (0 ppm).
For the synthesis of the 4-(methylsulfide)benzyl 2-diazoacetate,
the previous experimental procedure was employed. The expected
product was obtained as a yellow oil (yield 88%). 1H NMR (CDCl3,
400 MHz, ppm): ␦ 2.48 (s, 3H, SCH ), 4.79 (s, 1H, CH N ), 5.16 (s,
N
H
insertion of anilines in water/THF catalyzed by myo-
globin. Although, there are many examples showing that
,10,15,20-tetrakis-(4-sulfonato-phenyl)-porphyrin-iron(III) chlo-
5
3
2
2H, CH2), 7.24–7.30 (m, 4H, Ph). 13C NMR (CDCl , 125 MHz, ppm):
ride is a versatile water-soluble catalyst for oxidations in organic
syntheses [22] and few other reactions [23,24], its use for carbenoid
transfer in water seems to be neglected [25]. Comparative evalu-
ation of reactivity for N-H insertion of different diazo derivatives
with iron and ruthenium porphyrins will also be described.
3
␦ 15.85 (SCH ), 46.48 (CH N ), 66.28 (CH ), 126.68 (CHPh), 129.07
3
2
2
(CHPh), 132.72 (Cq), 139.08 (Cq), 166.8 (CO ). HR-MS (m/z): cal-
2
+
culated mass for C10H10N O NaS [M + Na] : 245.03607, found m/z:
2
2
245.0361 (0 ppm).
2.3. General procedure for NH insertion in water
2
. Experimental
In a typical experiment, the ␣-amino ester (0.18 mmol) and FeT-
2.1. General
SPPCl catalyst (1.8 ␮mol) were placed in a schlenck tube under
argon and dissolved in 2 ml of degazed bicarbonate buffer solu-
tion (pH 10). Ethyl diazoacetate (0.18 mmol) was then added at
room temperature. After 15 min of stirring the reaction was stopped
All reactions were performed under argon and were mag-
netically stirred. Solvents were distilled from appropriate drying
agent prior to use: MeOH from turning Mg. Commercially avail-
able reagents (Acros) were used without further purification unless
otherwise stated. All reactions were monitored by TLC with Merck
pre-coated aluminium foil sheets (Silica gel 60 with fluorescent
indicator UV254). Compounds were visualized with UV light at
and the mixture was then extracted three times with CH Cl , dried
2
2
by MgSO4 and purified by column chromatography on silica gel
CH Cl /Methanol:97/3). A similar procedure was effective for N
(
H
2
2
insertion catalyzed by RuTSPPCO, excepted that the reaction was
stopped after 4 h.
2
54 nm. Column chromatographies were carried out using silica
gel from Merck (0.063–0.200 mm). UV–vis spectra were recorded
on a UVIKON XL from Biotech. MALDI-TOF mass spectra were
recorded on microflex Bruker. The analysis were performed on
a ThermoFisher LC–MS system (a DionexUltiMate 3000 quater-
nary RSLC Nano-UPHLC system coupled with a Q-Exactive Hybrid
Quadrupole-Orbitrap Mass Spectrometer). Ethyl diazoacetate and
2.4. General procedure for NH insertion in organic solvent
In a typical experiment the ␣-amino ester (0.18 mmol) and
FeTPPCl (1.3 mg, 1.8 ␮mol) were placed in a schlenck tube under
argon and dissolved in 2 ml of distilled CH2Cl2. NEt3 (0.27 mmol)
was added to the solution. (Ethyl diazoacetate) (0.18 mmol) was
then added at room temperature. After 2 min of stirring, the inser-
tion product was purified by column chromatography on silica gel
(CH2Cl2/Methanol: 95/5).
5
,10,15,20-tetrakis-(4-sulfonato-phenyl)-porphyrin-iron(III) chlo-
ride are commercially available. 5,10,15,20-tetrakis-(4-sulfonato-
phenyl)-porphyrin-ruthenium(II) carbonyl was prepared as pre-
viously reported [14]. Diisopropyl diazomethylphosphonate was
prepared as previously reported [26,27].
2.5. NH insertion of insulin
2.2. Syntheses of diazo compounds
In a typical experiment, insulin (5 mg, 0.87 ␮mol) and 0.2 equiv
of TPPSFeCl (0.2 mg, 0.17 ␮mol) were dissolved in 10 ml of PBS and
ml of acetonitrile under nitrogen. 0.32 mg of cobaltocene was first
The synthesis of substituted benzyl diazoacetates was adapted
from the method previously reported by Fukuyama [28]. For
the synthesis of the 4-(trifluoromethyl)benzyl 2-diazoacetate,
-(trifluoromethyl)benzyl bromoacetate was first prepared: 4-
trifluoromethyl)benzyl alcohol (548 ␮L, 4.0 mmol) and NaHCO3
5
added to the solution and then, the diazo derivative (1.3 mmol)
diluted in 10 ␮l of acetonitrile. The solution was stirred for 1 h. The
yield was estimated by MALDI-TOF. After centrifugation, insulin
was purified by preparative HPLC and then dried under vacuum to
give a white powder.
4
(
(
1.0 g, 12.0 mmol) were dissolved in acetonitrile (20 ml) and bro-
◦
moacetylbromide (524 ␮l, 6.0 mmol) was added slowly at 0 C in a
1
00 ml round bottom flask. After stirring for 15 min at room tem-
perature, the reaction was quenched with H O. The solution was
2.6. Characterization of N H inserted compounds
2
extracted with CH Cl three times. The organic phase was washed
2
2
with brine and dried over MgSO . The solvent was evaporated,
2.6.1. Reaction of ethyl diazoacetate with amino esters
4
1
and the residue was used in the next reaction without purifica-
tion. The 4-(trifluoromethyl)benzyl bromoacetate thus obtained
Mono-inserted tyrosine methyl ester: H NMR (500 MHz, CDCl ,
3
ppm): ␦ 1.23 (t, J = 7.2 Hz, 3H, CH ), 2.88–2.98 (m, 2H, CH ), 3.37 (sys
3
2
ꢀ
and N,N -ditosylhydrazine (272 mg, 8.0 mmol) were dissolved in
AB, J = 17 Hz, 2H, CH ), 3.57 (t, 6.8 Hz, 1H, CH), 3.67 (s, 3H, OCH ),
2
3
◦
THF (20 ml) and cooled to 0 C. DBU (3 ml, 20 mmol) was added
4.14 (q, 7 Hz,2H, CH ), 6.71 (d, J = 8.5 Hz, 2H, CH), 7.02 (d, 8.5 Hz, 2H,
2
dropwise and stirred at room temperature for 10 min. After quench-
ing the reaction by the addition of saturated NaHCO3 solution, this
was extracted three times with diethylether. The organic phase
was washed with brine, dried over MgSO4 and evaporated to give
the crude diazo acetate. Purification of the diazo acetate was per-
formed with neutral silica gel to give 4-(trifluoromethyl)benzyl
CH). 13C NMR (125 MHz, CDCl , ppm): ␦ 14.13 (CH (CO Et)), 38.56
3
3
2
(CH Ph), 49.11 (CH NH), 51.92 (CH (CO Me)), 61.02 (CH (CO Et)),
2
2
3
2
2
2
62.23 (*CH), 115.51 (CH, Ph), 128.33 (Cq, CH Ph), 130.32 (CH, Ph),
2
154.9 (Cq (PhOH)), 171.75 (Cq (CO Et)), 174.22 (Cq (CO Me)). HR-
2
2
+
MS (m/z): calculated mass for C14H19NO5 [M + Na] : 304.29, found
m/z: 304.11(3 ppm)

1
96
H.F. Srour et al. / Journal of Molecular Catalysis A: Chemical 407 (2015) 194–203
Scheme 1. Synthesis of benzyl diazoacetate derivatives.
Bis-inserted tyrosine methyl ester: 1H NMR (500 MHz, CDCl3,
ppm): ␦ 1.25 (t, J = 7.2 Hz, 6H, 2xCH ), ␦ 2.91–2.99 (m, 2H, CH ),
4.14 (q, J = 7.1 Hz, 4H, 2xCH ), 7.17–7.27 (m, 5H, Ph). 13C NMR
2
(125 MHz, CDCl , ppm): ␦ 14.34 (CH3 (CO Et)), 37.28 (CH Ph),
3
2
3
2
2
␦
␦
8
3.57 (s, 3H, OCH ), 3.63 (t, 6.8 Hz, 1H, CH), 3.71 (s, 4H, 2xCH ),
52.87 (CH NH), 51.46 (CH3 (CO Me)), 60.75 (CH2 (CO Et)), 66.9
(*CH), 126.69 (CH, Ph), 128.50 (CH, Ph), 129.37 (CH, Ph), 137.50 (Cq
3
2
2 2 2
4.14 (q, 7 Hz, 2H, CH ), ␦ 6.71 (d, J = 8.5 Hz, 2H, CH), ␦ 7.02 (d,
2
1
3
.5 Hz, 2H, CH). C NMR (125 MHz, CDCl , ppm): ␦ 14.32 (CH
(PhCH )), 171.48 (Cq (CO Et)), 172.26 (Cq (CO Me)). HR-MS (m/z):
3
3
2
2
2
+
(
CO Et)), ␦ 36.37 (CH Ph), ␦ 51.50 (CH (CO Me)), ␦ 52.85 (CH N),
calculated mass for C18H25NO Na [M + Na] : 374.15796; found m/z:
2
2
3
2
2
6
*
␦
60.82 (CH2 (CO Et)), ␦ 67.12 ( CH), ␦ 115.38 (CH, Ph), ␦ 129.34
374.1590 (3 ppm).
2
(
Cq, CH Ph), ␦ 130.48 (CH, Ph), ␦ 154.56 (Cq (PhOH)), ␦ 171.60 (Cq
Mono-inserted glycine methyl ester: 1H NMR (500 MHz, CDCl3,
2
(
CO Et)), ␦ 172.47 (Cq) (CO Me). HR-MS (m/z): calculated mass for
ppm): ␦ 1.25 (t, J = 7.13 Hz, 3H, CH ) (CO Et), 3.44 (s, 2H, CH ), 3.46
2
2
3 2 2
+
C18H25NO7Na [M + Na] : 390.15287, found m/z: 390.1528 (0 ppm).
(s, 2H, CH (CH NH)), 3.71 (s, 3H, CH (CO Me)), 4.17 (q, J = 7.17 Hz,
2 2 3 2
1
13
Mono-inserted tryptophan methyl ester: H NMR (500 MHz,
2H, CH2 (CO Et)). C NMR (125 MHz, CDCl , ppm): ␦ 14.27 (CH3
2
3
CDCl , ppm): ␦ 1.19 (t, J = 7.1 Hz, 6H, CH ), 3.12–3,27 (m, 2H, CH ),
(CO Et)), 50.05 (CH ), 50.23 (CH ), 51.93 (CH ), 60.95 (CH (CO Et)),
2 2 2 3 2 2
3
3
2
3
1
.39 (sys AB, J = 17 Hz, 2H, CH ), 3.66 (s, 3H, OCH ), 3.70 (t, 6.1 Hz,
171.83 (Cq (CO Et)), 172.30 (Cq (CO Me)). HR-MS (m/z): calculated
2
3
2
2
+
H, CH), 4.10 (q, 7.1 Hz, 2H, CH ), 7.08–7.13 (m, 2H, CH), 7.18 (t,
mass for C7H13NO Na [M + Na] : 198,07423; found m/z: 1,980,748
2
4
J = 7.3 Hz, 1H, CH), 7.34 (d, J = 8 Hz, 1H, CH), 7.6 (d, J = 7.8 Hz, 1H, CH),
(3 ppm).
␦
8.19 (s, 1H, NH (indole)). 13C NMR (125 MHz, CDCl , ppm): ␦ 14.25
Bis-inserted glycine methyl ester: 1H NMR (500 MHz, CDCl3,
3
(
CH (CO Et)), 29.27 (CH Ph), 49.40 (CH N), 52.04 (CH (CO Me)),
ppm):
␦ 1.24 (t, J = 7.2 Hz, 3H, CH3) (CO Et), 3.63 (s, 4H,
3
2
2
2
3
2
2
6
0.97 (CH2 (CO Et)), 61.32 (*CH), 111.03 (Cq), 111.32 (CH), 118.83
CH (NHCH )), 3.65 (s, 2H, CH (CH NH)), 3.69 (s, 3H, CH (CO Me)),
4.15 (q, J = 7.2 Hz, 2H, CH (CO Et)). C NMR (125 MHz, CDCl3,
2 2
2
2
2
2
2
3
2
13
(
(
CH), 119.63 (CH), 122.27 (CH), 123.08 (CH), 127.54 (Cq), 136.35
Cq), 171.78 (Cq (CO Et)), 174.52 (Cq (CO Me)). HR-MS (m/z): cal-
ppm): ␦ 14.31 (CH3 (CO Et)), 51.81 (CH3 (CO Me)), 55.10 (CH2
2
2
+
2
2
culated mass for C16H20N O Na [M + Na] : 327.13208; found m/z:
(CH CO Me)), 55.29 (NHCH ), 60.80 (CH (CO Et)), 170.87 (Cq
2 2 2 2 2
2
4
3
27.1322.
Bis-inserted tryptophan methyl ester: H NMR (500 MHz, CDCl ,
(CO Et)), 171.37 (Cq (CO Me)). HR-MS (m/z): calculated mass
2
2
1
+
for C11H19NO Na [M + Na] : 284.11101; found m/z: 284.11111
3
6
ppm): ␦ 1.25 (t, J = 7.1 Hz, 6H, 2xCH ), ␦ 3.13–3.28 (m, 2H, CH ),
(0 ppm).
3
2
␦
␦
3.57 (s, 3H, OCH ), 3.76 (s, 4H, 2xCH ), 3.82 (t, 7.4 Hz,1H, CH),
Mono-inserted lysine methyl ester: 1H NMR (500 MHz, CDCl3,
3
2
4.14 (q, J = 7 Hz, 2H, CH ), ␦ 7.11 (t, J = 7.3 Hz, 1H, CH), 7.17 (t,
ppm): ␦ 1.23 (t, 3H, CH ), 1.45 (m, 4H, 2xCH ), 1.65 (m, 2H, CH ),
2
3 2 2
J = 7.2 Hz, 2H, CH), 7.33 (d, J = 8 Hz, 1H, CH), 7.63(d, J = 7.8 Hz, 1H,
2.69 (t, 2H, CH ), 3.36 (sys AB, 17.0 Hz, 2H, CH NH), 3.71 (s, 3H,
2
2
CH), ␦ 7.99 (s, 1H, NH (indole)). 13C NMR (125 MHz, CDCl , ppm):
CH ), 4.15 (q, 2H, CH ), 4.17 (m, 1H, CH).
3
3
2
␦
5
␦
14.34 (CH3 (CO Et)), ␦ 26.58 (CH Ph), ␦ 51.45 (CH (CO Me)), ␦
Mono-inserted S-H cysteine ethyl ester: 1H NMR (400 MHz,
CDCl , ppm): ␦ 1.32 (2t, 6H, CH ), 2.97–3.13 (m, 2H, CH C*), 3.26
2
2
3
2
2.78 (CH N), ␦ 60.73 (CH2 (CO Et)), ␦ 65.62 (*CH), ␦ 111.16 (CH),
2
2
3
3
2
111.45 (Cq), ␦ 118.79 (CH), ␦ 119.51 (CH), ␦ 122.06 (CH), ␦ 123.15
(s, 2H, CH S), 3.79 (m, 1H, C*H), 4.24 (2m, 4H, CH ) containing also
2
2
(
(
[
CH), ␦ 127.61 (Cq), ␦ 136.15 (Cq), ␦ 171.61 (Cq (CO Et)), ␦ 172.76
20% of N-H inserted cysteine.
2
Cq) (CO Me). HR-MS (m/z): calculated mass for C20H26N O Na
2
2
6
+
M + Na] : 413.16886; found m/z: 413.1701.
Mono-inserted phenylalanine methyl ester: 1H NMR (500 MHz,
2.6.2. Reaction of 4-(trifluoromethyl)benzyl 2-diazoacetate with
amino esters
CDCl , ppm): ␦ 1.23 (t, J = 7.2 Hz, 3H, CH ), 2.94–3.05 (m, 2H, CH ),
3
3
3
3
2
Mono-inserted phenylalanine methyl ester: 1H NMR (500 MHz,
.36 (sys AB, J = 17 Hz, 2H, CH NH), 3.60 (t, J = 6.7 Hz,1H, CH), 3.66 (s,
2
13
CDCl3, ppm): ␦ 2.93–3.05 (m, 2H, CH2), 3.42 (sys AB, J = 17.40 Hz,
H, CH ), ␦ 4.14 (q, J = 7.1 Hz, 2H, CH ), ␦ 7.19–7.30 (m, 5H, Ph).
C
3
2
*
2
H, CH NH), 3.61 (t, J = 6.90 Hz, 1H, CH), 3.65 (s, 3H, OCH ), 5.16
2
3
NMR (125 MHz, CDCl , ppm): ␦ 14.13 (CH (CO Et)), 39.67 (CH Ph),
3
3
2
2
(
s, 2H, OCH ), 7.17–7.29 (m, 4H, Ph), 7.42 (d, J = 8 Hz, 2H, Ph), 7.60
2
4
9.32 (CH NH), 51.85 (CH3 (CO Me)), 60.99 (CH2 (CO Et)), 62.31
2 2 2
13
*
(d, J = 8 Hz, 2H, Ph). C NMR (125 MHz, CDCl3, ppm): ␦ 39.67 (CH2),
(
(
CH), 127 (CH, Ph), 128.65 (CH, Ph), 129.32 (CH, Ph), 137.04 (Cq
*
4
9.27 (CH ), ␦ 51.99 (CH O), 62.23 ( CH), 65.71 (CH ), 124.09 (q,
2
3
2
PhCH )), 171.72 (Cq (CO Et)), 174.21 (Cq) (CO Me). HR-MS (m/z):
2
2
2
1
3
+
JCF = 272.14 Hz, CF3), 125.68 (q, JCF = 3,78 Hz, Ph), 127.04 (Ph),
calculated mass for C14H19NO5Na [M + Na] : 288,12118; found m/z:
2
2
1
28.40 (Ph), 128.66 (Ph), 129.31 (Ph), 130.66 (q, JCF = 32.57 Hz, Cq),
88,1207 (2 ppm).
Bis-inserted phenylalanine methyl ester: 1H NMR (500 MHz,
136.95 (Cq), 139.59 (Cq), 171.45 (Cq, CO2CH2), 174.13 (Cq, CO2Me).
NMR 19F (CDCl3, 376 MHz, ppm): ␦ −62.685. HR-MS (m/z): calcu-
CDCl , ppm): ␦ 1.25 (t, J = 7.1 Hz, 6H, 2xCH ), 2.99–3.08 (m, 2H,
CH ), 3.57 (s, 3H, CH ), 3.66–3.70 (m, 1H, CH), 3.72 (s, 4H, 2xCH ),
3
3
+
lated mass for C20
H
20
NO F Na [M + Na] : 41,812,421; found m/z:
4 3
2
3
2
4
,181,239 (1 ppm).

H.F. Srour et al. / Journal of Molecular Catalysis A: Chemical 407 (2015) 194–203
197
Mono-inserted tryptophan methyl ester: 1H NMR (500 MHz,
3.69 (t, J = 6.08 Hz, 1H, CH), 5.03 (sys AB, J = 5.12 Hz, 2H, CH2Ph),
7.09 (d, J = 2.02 Hz, 1H, Hindole), 7.12 (t, J = 7.78 Hz, 1H, Hindole),
7.18 (t, J = 7.29 Hz, 1H, Hindole), 7.35 (d, J = 8.03 Hz, 1H, Hindole),
7.6 (d, J = 7.84 Hz, 1H, Hindole), 8.07 (s, 1H, NHindole). 13C NMR
(125 MHz, CDCl3, ppm): ␦ 15.73 (CH3S), 29.21 (CH2), 49.35 (CH2),
52.0 (CH3O), 61.23 (*CH), 66.29 (CH2), 110.71 (Cq), 111.35 (CH),
118.69 (CH), 119.48 (CH), 122.11 (CH), 123.19 (CH), 126.53 (CH),
127.43 (Cq), 129.1 (CH), 132.23 (Cq), 136.3 (Cq), 139.06 (Cq), 171.66
(Cq, COOCH2), 174.48 (Cq, CO2Me). HR-MS (m/z): calculated mass
CDCl , ppm): ␦ 2.07 (s, 1H, NH), ␦ 3.12–3.28 (m, 2H, CH ), 3.48 (sys
3
2
AB, J = 17.38 Hz, 2H, CH -NH), 3.66 (s, 3H, CH ), 3.72 (t, J = 7.08 Hz,
2
3
1
1
H, CH), 5.12 (sys AB, J = 6.82 Hz, 2H, CH Ph), 7.07 (d, J = 1.99 Hz,
2
H, Hindole), 7.13 (t, J = 7.75 Hz, 1H, Hindole), 7.2 (t, J = 7.69 Hz, 1H,
Hindole), 7.34 (d, J = 8.11 Hz, 1H, Hindole), 7.38 (d, J = 8.01 Hz, 2H,
Ph), 7.59 (d, J = 7.97 MHz, 2H, Ph), 7.60 (d, J = 7.04 Hz, 1H, Hindole),
␦
8.12 (s, 1H, NHindole). 13C NMR (125 MHz, CDCl , ppm): ␦ 29.27
3
(
(
(
1
(
CH ), 49.34 (CH ), 52.05 (OCH ), 61.26 (*CH), 65.63 (CH ), 110.86
2 2 3 2
+
Cq), 111.37 (CHNH), 118.75 (Ph), 119.59 (Ph), 122.24 (Ph), 123.15
for C22H24N2O4NaS [M + Na] : 435.13545; found m/z: 435.1357
3
Ph), 124.08 (q, 1JCF = 272.04 Hz, CF ), 125.61 (q, JCF = 3.74 Hz, CH),
(1 ppm).
3
1
27.47 (Cq), ␦ 128.33 (Ph), 130.54 (q, 2JCF = 32.42, Cq), 136.36
Mono-inserted tyrosine methyl ester: H NMR (500 MHz, CDCl3,
19
Cq), 139.57 (Cq), 171.54 (Cq, CO CH ), 174.48 (Cq, CO Me).
F
ppm): ␦ 2.51 (s, 3H, CH3S), 2.9–3.02 (m, 2H, CH2), 3.45 (AB sys,
J = 16.48 Hz, 2H, CH2NH), 3.61 (t, J = 6.72 Hz, 1H, CH), 3.7 (s, 3H,
CH3O), 5.10 (s, 2H, CH2), 6.72 (d, J = 8.37 Hz, 2H, PhOH), ␦ 7.04
(d, J = 8.33 Hz, 2H, PhOH), ␦ 7.24–7.28 (m, 4H, PhSMe). 13C NMR
(125 MHz, CDCl3, ppm): ␦ 15.73 (CH3S), 38.59 (CH2), 49.17 (CH2),
52.05 (CH3O), 62.24 (C*H), 66.51 (CH2), 115.65 (Ph), 126.57 (Ph),
128.13 (Cq), 129.14 (Ph), 130.37 (Ph), 132.1 (Cq), 139.14 (Cq), ␦
155.13 (Cq), 171.68 (Cq, CO2CH2), 174.3 (Cq, COOMe). HR-MS (m/z):
2
2
2
NMR (CDCl , 376 MHz, ppm): ␦ −62.632. HR-MS (m/z): calculated
3
+
mass for C
H
N O F Na [M + Na] : 457.1351; found m/z: 457.135
2
2
21
2
4 3
(
0 ppm).
Mono-inserted tyrosine methyl ester: H NMR (500 MHz, CDCl ,
1
3
ppm): ␦ 2.87–2.96 (m, 2H, CH ), 3.46 (sys AB, J = 17.4 Hz, 2H,
2
CH NH), 3.59 (t, J = 6.60 Hz, 1H, CH), 3.67 (s, 3H, CH ), ␦ 5.16 (s,
2
3
2
2
H, CH OCO), 6.69 (d, J = 8.44, Hz, 2H, PhOH), 7.0 (d, J = 8.44 Hz,
2
+
H PhOH),), 7.4 (d, J = 8.03 Hz, 2H PhCF ), 7.6 (d, J = 8.1 Hz, 2H
calculated mass for C20H23NO5NaS [M + Na] : [412.11946]; found
3
1
3
PhCF ). C NMR (125 MHz, CDCl , ppm): ␦ 38.59 (CH ), 49.11
m/z: 412.1195 (0 ppm).
3
3
2
(
(
1
CH ), 52.1 (CH O), 62.23 (*CH), 65.84 (CH ), 115.68 (Ph), 124.05
2 3 2
1
3
q, JCF = 272.08 Hz, CF ), 125.65 (q, JCF = 3.71 Hz, CH), 128.12 (Cq),
3
2
28.37 (Ph), 130.40 (Ph), 130.45 (q, JCF = 32.51 Hz, Cq), 139.42 (Cq),
55.15 (Cq), 171.56 (Cq, CO CH ), 174.32 (Cq, CO Me). F NMR
2.6.4. Reaction of diisopropyl diazomethylphosphonate with
amino acid derivatives
19
1
2
2
2
Mono-inserted aniline: 1H NMR (500 MHz, CDCl3, ppm): ␦ 1.32
(d, J = 6.2 Hz, 6H, 2xCH3), 1.37 (d, J = 6.2 Hz, 6H, 2xCH3), 3.46 (d,
JHP = 13.7 Hz, 2H, CH2), 3.96 (s broad, 1H, NH), 4.73–4.82 (m,
JHH = JHP = 6.3 Hz, 2H, CHOP), 6.68 (d, J = 7.8 Hz, 2H, Ph), 6.77 (t,
(
CDCl , 376 MHz, ppm): ␦ −62.668. HR-MS (m/z): calculated mass
3
+
for C
H
20
NO5F Na [M + Na] : 434.11913; found m/z: 434.1187
2
0
3
(
1 ppm).
Mono-inserted phenylalanine methyl ester: 1H NMR (500 MHz,
CDCl , ppm): ␦ 4.00 (s, 2H, CH , (NHCH )), ␦ 5.26 (s, 2H, CH ), 6.62
13
J = 7.3 Hz, 1H, Ph), 7.21 (t, J = 7.5 Hz, 2H, Ph).
C NMR, HMBC
3
2
2
2
3
(
d, J = 7.71 Hz, 2H, o-Ph), ␦ 6.77 (t, J = 7.34 Hz, 1H, p-Ph), 7.20 (t, 2H,
(125 MHz, CDCl3, ppm): ␦ 23.94 (d, JCP = 4.7 Hz, 2xCH3), 24.06
(d, JCP = 3.8 Hz, 2xCH3), 41.03 (d, 1JCP = 156.13 Hz, NHCH2), 71.18
3
J = 7.44 Hz, m-Ph), 7.42 (d, J = 8.01 Hz, 2H, Ph), 7.62 (d, J = 8.01 Hz,
2
1
3
2
H, Ph). C NMR (125 MHz, CDCl , ppm): ␦ 46.02 (CH ), 66.10
(d, JCP = 6.72 Hz, 2xCHOP), 113.26 (o-Ph), 118.37 (p-Ph), 129.19
3
2
1
3
31
(
CH ), 113.19 (CH), 118.70 (CH), 124.07 (q, JCF = 272.17 Hz, CF ),
(m-Ph), 147.67 (d, JCP = 3.8 Hz, Cq). P NMR (162 MHz, CDCl3,
ppm): ␦ 22.68 (s). HRMS (m/z): calculated mass for C13H22NO3NaP
2
3
2
1
25.72 (q, JCF = 3.72 Hz, CH), 128.41 (Ph), 129.50 (Ph), 130.73 (q,
2
+
JCF = 32.53 Hz, Cq), 139.41 (Cq), 146.99 (Cq), 171.12 (Cq, CO CH ).
(M + Na) : 294.1235; found m/z: 294.1236 (0 ppm).
2
2
1
9
Mono-inserted tryptophan methyl ester: 1H NMR (500 MHz,
CDCl3, ppm): ␦ 1.22 (t, J = 6.44 Hz, 6H, 2xCH3), ␦ 1.26 (t, J = 6.59 Hz,
6H, 2xCH3), ␦ 2.79 (t, J = 14.37 Hz, 1H, CH2), 3.04 (t, J = 12.94 Hz, 1H,
CH ), 3.08–3.21 (m, 2H, CH (tryp)), 3.63 (s, 3H, CH (OCH )), 3.73 (t,
NMR F (CDCl , 376 MHz, ppm): ␦ −62.682. HR-MS (m/z): calcu-
3
+
lated mass for C
H
14
NO F Na [M + Na] : 332.08743; found m/z:
1
6
2 3
3
32.0873 (0 ppm).
2
2
3
3
J = 6.39 Hz, 1H, *CH), 4.6–4.7 (m, JHH = JHP = 6.3 Hz, 2H, CHOP), 6.99
(s, 1H, Hindole), 7.08 (t, 1H, 7.38 Hz, Ph), 7.15 (t, 1H, J = 7.76 Hz,
Ph), 7.33 (d, 1H, J = 8.1 Hz, Ph), 7.57 (d, 1H, 7.88 Hz, Ph). 13C NMR
(125 MHz, CDCl , ppm): ␦ 23.96 (2d, J = 5.09, 5.78 Hz, 2xCH ), 24.12
2
.6.3. Reaction of 4-(methylsulfide)benzyl 2-diazoacetate with
amino acid derivatives
Mono-inserted aniline: 1H NMR (500 MHz, CDCl , ppm): ␦ 2.38
3
3
3
1
(
s, 3H, (SCH )), 3.83 (s, 2H, (CH ) NHCH ), 5.06 (s, 2H, CH Ph),
(d, J = 3.74 Hz, 2xCH ), 29.13 (CH , (tryp)), 44.47 (d, J = 158.17 Hz,
3
2
2
2
3 2 CP
3
6
2
.50 (d, J = 7.69 Hz, 2H,o-Ph), 6.65 (t, J = 7.34 Hz, 1H, p-Ph), 7.08 (t,
NHCH ), 51.86 (OCH ), 62.82 (d, JCP = 15.44 Hz, *CH), 70.78 (d,
2 3
H, J = 7.42 Hz, o-Ph), 7.15 (sys AB, J = 8.65 Hz, 4H, Ph). 13C NMR
2
J = 1.8 Hz, CH), 70.94 (s, CH), 110.68 (Cq indole), 111.38 (CH indole),
(
1
1
125 MHz, CDCl , ppm): ␦ 15.71 (SCH ), 45.98 (CH ), 66.76 (CH ),
13.11 (CH), 118.37 (CH), 126.57 (CH), 129.16 (CH), 129.40 (CH),
118.71 (CH indole), 119.35 (CH indole), 121.95 (CH indole), 123.20
(CH indole), 127.54 (Cq indole), 136.39 (Cq indole), 174.55 (Cq
3
3
2
2
32.05 (Cq), 139.31 (Cq), 147.03 (Cq), 171.15 (Cq). HR-MS (m/z): cal-
(CO)).31P NMR (162 MHz, CDCl , ppm): ␦ 23.13 (s). HR-MS (m/z):
3
+
+
culated mass for C16H17NO NaS [M + Na] : 310.08777; found m/z:
calculated mass for (C19H29N O5NaP) [M + Na] : 419.1711; found:
2
2
3
10.0879 (0 ppm).
419.1712 (0 ppm).
Mono-inserted phenylalanine methyl ester: 1H NMR (500 MHz,
Mono-inserted tyrosine methyl ester: H NMR (500 MHz, CDCl ,
1
3
CDCl , ppm): ␦ 2.01 (s, 1H, NH), 2.5 (s, 3H, CH S), 2.96–3.08 (m,
ppm): ␦ 1.22 (t, J = 5.81 Hz, 6H, CH ), 1.26 (d, J = 6.18 Hz, 6H,
3
3
3
2
1
H, CH ), 3.43 (sys AB, J = 16.34 Hz, 2H, CH NH), 3.64 (t, J = 6.81 Hz,
CH ), 2.74 (t, J = 14.38 Hz, 1H, CH ), 2.77–2.93 (m, 2H, CH (tyro-
sine)), 3.01 (t, J = 11.85 Hz, 1H, CH ), 3.59 (t, J = 6.58 Hz, 1H, *CH),
2
2
2
3
2
2
H, CH), 3.67 (s, 3H, CH O), 5.1 (s, 2H, CH O), 7.2–7.31 (m, 9H,
3
2
13
Ph). C NMR (125 MHz, CDCl , ppm): ␦ 15.78 (CH S), 39.57 (CH ),
3.65 (s, 3H, OCH ), 4.58–4.69 (m, JHP = J = 6.01 Hz, 2H, CHOP),
3
3
2
3 CP
13
4
1
1
9.24 (CH ), 51.89 (CH O), 62.17 (*CH), 66.32 (CH ), 123.90 (Ph),
6.74 (d, 2H, 8.33 Hz, Ph), 6.96 (d, 1H, 8.33 Hz, CH (Ph). C NMR
2
3
2
3
26.60 (Ph), 126.94 (Ph), 128.58 (Ph), 129.120 (Ph), 129.26 (Ph),
(125 MHz, CDCl , ppm): ␦ 23.96 (2d, JCP = 4.86 Hz, 2xCH ), ␦
3
3
3
32.26 (Cq), 136.94 (Cq), 139.13 (Cq), 171.52 (Cq, CO CH2), 174.08
24.1 (d, JCP = 3.63 Hz, CH3), 38.67 (CH2 (tyrosine)), 44.32 (d,
2
1
3
(
[
Cq, CO Me). HR-MS (m/z): calculated mass for C20H23NO NaS
M + Na] : [M + Na]+: 396.12455; found m/z: 396.1248 (1 ppm).
JCP = 160.04 Hz, NHCH ), 51.86 (OCH3)), 63.71 (d, JCP = 14.96 Hz,
2
+
4
2
2
*CH), 71.42 (2d, JCP = 6.84 Hz, CH) 115.58 (Ph), 127.54 (Cq, Ph),
1
31
Mono-inserted tryptophan methyl ester: H NMR (500 MHz,
130.24 (Ph), 156.1 (Cq), 174.41 (Cq (CO Me)). P NMR (162 MHz,
2
CDCl , ppm): ␦ 1.93 (s, 1H, NH), 2.47 (s, 3H, CH S), 3.15–3,27 (m,
CDCl , ppm): ␦ 22.93 (s). HR-MS (m/z): calculated mass for
3
3
3
+
2
H, CH ), 3.44 (sys AB, J = 16.35 Hz, 2H, CH NH), 3.64 (s, 3H, CH O),
(C17H28NO NaP) [M + Na] : 396.1552; found: 396.1553 (0 ppm).
2
2
3
6

1
98
H.F. Srour et al. / Journal of Molecular Catalysis A: Chemical 407 (2015) 194–203
Fig. 1. Structure of the catalysts.
Mono-inserted phenylalanine methyl ester: 1H NMR (500 MHz,
CDCl , ppm): ␦ 1.24 (t, J = 7.16 Hz, 6H, 2xCH ), 1.27–1.29 (m,
5,10,15,20-tetrakis-(4-sulfonato-phenyl)-porphyrin-iron(III) chlo-
ride (FeTSPPCl) (Fig. 1) in water.
3
3
6
2
1
H, 2xCH ), 1.71 (s large, 1H, NH), 2.76 (t, 1H, CH2 (NHCH )),
The results are summarized in Table 1. Thus, FeTSPPCl catalyzed
decomposition of 1 equiv of ethyl diazoacetate in the presence of
tyrosine methyl ester in dichloromethane resulted in the forma-
tion of mono N-alkylated amino esters in 71% yield, containing 14%
of bis-inserted compound (Table 1, entry 4). A similar result was
obtained for tryptophan with 66% yield (Table 1, entry 3). In this
case 18% of insertion was the bis-inserted compound (Scheme 2).
To extend the scope of this N H insertion, the reaction of a number
of amino acid derivatives with ethyl diazoacetate in the pres-
ence of TSPPFeCl as catalyst was studied at room temperature
3
2
.89–3.02 (m, CH2 (Phenylala.) +1CH2 (NHCH )), 3.64 (t, 6.77 Hz,
2
H, *CH), 3.67 (s, 3H, (OCH )), 4.61–4.70 (m, 2H, CH, 7.17–7.28
3
1
3
(m, 5H, Ph)).
C NMR (125 MHz, CDCl , ppm): ␦ 24.06 (2d,
3
3
3
JCP = 4.9 Hz, 2xCH3(isopropyl)), 24.2 (2d, 3JCP = 2.57 Hz, 2xCH ),
9.56 (CH (phenylala.)), 45.29 (CH , NHCH ), 51.88 (OCH )), 63.66
3
2
2
2
3
2
(
1
d, 3JCP = 15.42 Hz, *CH), 70.91 (2d), JCP = 6.52, 6.93 Hz, 2xCH,
26.88 (Ph), 128.56 (Ph), 129.36 (Ph), 137.23 (Cq), 174.28 (Cq
3
1
(
CO Me)). NMR P (162 MHz, CDCl , ppm): 22.73 (s). HR-MS (m/z):
2
3
+
calculated mass for C17H28NO5PNa [M + Na] : 380.16028; found
m/z: 380.1598 (1 ppm).
◦
(25 C). In all these experiments, N-H insertion is the major reac-
tion yielding alkylated products with moderate yield (43–71%),
usually obtained with a small amount of bis-inserted compounds.
Glycine is an exception since, in this case, the bis-inserted com-
pounds was obtained with 32% yield (Table 1, entry 6). A complete
regioselectivity is obtained with tyrosine, since no O H insertion
was detected. With tryptophan methyl ester, only ␣-amino group
was reactive since no insertion was detected in the NH group of
indole ring. Because the difference of pKa values of the N-terminal
group (∼7.6–8) and the NH2 group in the side chain of lysine
residue (10.0–10.2), this reaction (81% yield) can also be optimized
to maximize the selectivity for the N-terminal insertion by the
reaction being performed under weakly basic conditions (pH 9).
Actually cysteine ethyl ester was also tested to detect any selec-
tively. With this amino ester, both N H insertion and S H insertion
3
. Results and discussion
3.1. Synthesis of diazo derivatives
Besides ethyl diazoacetate and diisopropyl diazomethylphos-
phonate, two substituted benzyl diazo acetates were also
investigated in order to introduce SMe and trifluoromethyl groups.
A tosyl group was employed for the synthesis due the availabil-
ity of inexpensive p-toluenesulfonyl hydrazide. The first step
was the preparation of 4-(trifluoromethyl)benzylbromoacetate
and
4-(methylsulfide)benzylbromoacetate
from
4-
(
trifluoromethyl)benzyl alcohol and 4-(methylsulfide)benzyl
alcohol, respectively. Finally, these bromoacetates underwent an
easy conversion to the corresponding diazo acetates upon treat-
ment with N,N -ditosylhydrazine and DBU at 0 C, following the
protocol previously reported by Fukuyama [28]. The preparation
of 4-(trifluoromethyl)benzyl 2-diazoacetate proceeded in a 90%
yield, while that of 4-(methylsulfide)benzyl 2-diazoacetate in 88%
ꢀ
◦
Table 1
Ethyl diazoacetate insertion in N H bonds of amino esters catalyzed by FeTSPPCl
a
and RuTPPSCO in water.
Catalyst
Substrate
Time
Yield
Mono/bis
ratio (%)b
1
yield. These diazoacetates were characterized by H and the latter
19
by F NMR spectroscopy. We note the presence of a characteristic
peak at 4.83 ppm and 4.79 ppm (broad singlet) corresponding to
CH neighboring N2 for the CF3 and SCH3 derivatives, respectively.
1
2
3
4
5
6
7
8
9
–
R = tryptophan
Aniline
24
0
–
TSPPFeCl
TSPPFeCl
TSPPFeCl
TSPPFeCl
TSPPFeCl
TPPFeCl
TPPFeCl
TSPPFeCl
TSPPFeCl
TSPPRuCO
15 min
15 min
15 min
15 min
15 min
2 min
2 min
24 h
43
66
71
62
43
82
80
Traces
48
56
100:0
82:18
86:14
83:17
68:32
90:10
89:11
R = tryptophan
R = −CH2PhOH
R = −CH2Ph
c
R = H
3.2. NH insertion of amino acid esters by ethyl diazoacetate in
R = tryptophan
R = −CH2Ph
water
Mono-inserted tryp.
Mono-inserted tryp.
R = tryptophan
d
The insertion of electrophilic carbenes into the N H bonds of ␣-
10
1 h
4 h
1
1e
80:20
amino acids and ␣-amino acid esters could be a powerful method
for N-alkylating this important class of compounds. However there
are several drawbacks and the regioselectivity of the reaction still
appears as a difficult problem to solve. This is due to numerous
possible reactive sites such as acidic groups, amino groups, thiol
groups, and phenol groups. To shine some light on this aspect, we
first investigated the regioselectivity of the reaction catalyzed by
Reaction conditions:
a
◦
Catalyst/substrate/diazo mixture (1:100:100) in 1 ml of CBS (pH 10) at 25 C.
determined by H NMR.
Solvent: EtOH/CBS (50/50) (pH 10).
b
c
1
d
e
20 equiv of sodium dithionite vs catalyst are added.
Amino ester and diazo dissolved in ethanol are progressively added to a buffer
solution (CBS pH 10) containing the catalyst.

H.F. Srour et al. / Journal of Molecular Catalysis A: Chemical 407 (2015) 194–203
199
Scheme 2. Catalytic insertion of ethyl diazoacetate in N H bond of amino esters.
Fig. 2. 1H-13C HMBC spectrum of ethyl diazoacetate insertion in N H terminus bond of tryptophan.
1
were observed, the latter being largely predominant (>70%, from H
NMR) using one equivalent of the diazo derivative. Unfortunately,
we were not able to separate the two compounds.
Although, there are a large range of strategies and reagents for
coupling amino acids available, racemization of amino acids is still
a major problem in these reactions [29]. Due to the basic pH of the
carbonate buffer solution, it was necessary to verify the absence of
racemization during the insertion process [29,30]. Phenylalanine
methyl ester was used as substrate to check a possible racemiza-
tion. After ethyl diazoacetate coupling, the enantiomeric and the
racemic forms of phenylalanine were analyzed separately on HPLC
column with chiral phase as the stationary phase. The results are
summarized in Fig. 3. The chiral analysis gave 50% of each iso-
mer after insertion with racemic phenylalanine whereas a pure
enantiomer was obtained with L-phenylalanine, as expected for a
reaction without any racemization.
The diazo acetates were added to the amino acids in a
water–methanol solution containing the catalyst under argon.
Argon was used as a protecting atmosphere due to a possible for-
mation of reduced iron(II) in the catalytic cycle [17]. The reactions
were followed by TLC and were left until the complete disap-
pearance of the diazo acetates. We note that traces of the amino
acids remained when the reactions were over and this is due to
the formation of carbene dimers. The products were character-
ized by NMR spectroscopy. We note in the 1H NMR spectrum,
the presence of characteristic AB systems (∼3.4 ppm), which are
due to the two protons on the carbon next to the amine func-
tion. For example, the spectrum resulting after diazo insertion
in tryptophan is shown Fig. 2. This result was observed for all
chiral amino esters and is related to the presence of the asym-
metric carbon atom. With glycine, only one singlet was noted at
3
.3. NH insertion of amino acid esters by benzyl diazocetate
derivatives
We first investigated the reaction of 4-(thiomethyl)benzyl dia-
zoacetate and 4-(trifluoromethyl)benzyl diazoacetate with aniline
3
.6 ppm.
5
4
3
2
00
2
1
1
00
50
00
00
00
00
5
0
100
0
0
-22
-46
25
50
75
25
50
75
100
Mi nutes
Minutes
Fig. 3. HPLC chromatograms of racemic (A) and optically active (B) phenylalanine N H insertion with ethyl diazoacetate.

2
00
H.F. Srour et al. / Journal of Molecular Catalysis A: Chemical 407 (2015) 194–203
Scheme 3. Catalytic insertion of benzyl diazoacetate derivatives in N H bond of amino esters.
Scheme 4. Catalytic insertion of diisopropyl diazomethylphosphonate derivatives in N H bond of amino esters.
and amino esters (Table 2) in organic solvent. In particular, we
wanted to check if these reactions can tolerate the presence of sul-
fur atom in the diazo compound. The insertion reactions are shown
in Scheme 3 and the results reported in Table 2. In these case, the
catalyst used for these reaction was TPPFeCl (Fig. 1). The reactions
are very fast (<2 mn). The yields obtained are quite high (87–88%)
with the diazoester bearing the CF3 groups if we except the results
obtained with tyrosine (66%) (Table 2, entry 4) due probably to
␣-amino group was only observed with lysine methyl ester. As both
ruthenium and iron porphyrins react with nitrogen compounds to
form stable adducts, their decreased activity (by comparison with
cyclopropanation reactions) [31] can be attributed to competitive
coordination between the amino substrate and ethyl diazoacetate
onto the metal. Since, axial Ru N bond seems much stronger than
Fe N bond, iron catalysts are more efficient but less tolerant to
functional group such as thiol groups.
solubility difficulty in CH Cl . Aniline gave also a good yield (97%)
In order to give a clearer characterization of the products, a
HMBC spectrum was done for the NH insertion reaction of tyro-
sine with 4-(methylsulfide)benzyl 2-diazoacetate. This spectrum
shows the coupling between a carbon and hydrogen present on the
neighboring carbon. We note a coupling between the protons of
2
2
(Table 2, entry 1). In contrast, the yields are only moderate with
the diazo bearing the SMe group (50–67%) and some amount of the
starting material was recovered (Table 2, entries 5–8). A possible
interaction of the sulfur atom with iron may inhibit the catalyst,
and consequently may explain the low conversion. As previously
indicated with ethyl diazoacetate, all the insertion products were
CH2 (CH NH) and the asymmetric carbon of the amino acid. Also,
2
these protons are coupled to the carbon of the ester function. This
result proves the formation of the NH CH2 bond.
characterized by NMR, showing also an AB system for CH group in
2
alpha position of the nitrogen.
To examine whether these diazo derivatives could be used
for intermolecular N H insertion of amino acid derivatives in
protein, the catalytic reaction was also realized with water sol-
3
.4. NH insertion of amino acid esters by diisopropyl
diazomethylphosphonate
uble tetra-p-(SO -)phenyl metalloporphyrins (Fe and Ru). These
3
Intramolecular
N H insertion of diazomethylphosphonate
two metalloporphyrins were chosen because they are easily pre-
pared and commercially available. With the goal of developing
protein–compatible reaction conditions, we selected methanol as
a co-solvent for diazo ester and amino acid derivative solubilities.
All the reactions with benzyl diazoacetate derivatives are summa-
rized in Table 3. First, it should be noted that the yields are only
moderare (40–60%) using a small excess of the diazo derivatives
derivatives was first developed for the synthesis of antibiotic
carbapenems [32]. Since then Aller et al. [33] have developed signif-
icant diazomethylphosphonate-insertion reactions using rhodium
complexes as catalysts for a carbenoid approach to peptide synthe-
sis [34]. Many other biological applications have been related to the
use of N-(phophomethyl) aminoacids such as shikimate pathway
inhibitors [35] and selective thrombin inhibitors [36].
(
2 equiv) vs substrate. In these water conditions, insertion of the
Table 3
Table 2
Insertion of 4-(trifluoromethyl)benzyl diazoacetate (F) and 4-(thiomethyl)benzyl
Insertion of 4-(trifluoromethyl)benzyl diazoacetate (F) and 4-(thiomethyl)benzyl
diazoacetate (S) in N H bond of amino esters catalyzed by TPPFeCl in CH2Cl2.
diazoacetate (S) in
water/methanol.
N H bond of aminoesters catalyzed by TPPSFeCl in
Diazo
Catalyst
Substrate
Time
Yield (%)a
Diazo
Catalyst
Amino ester
Time
Yield (%)
1
2
3
4
5
6
7
8
F
F
F
F
S
S
S
S
TPPFeCl
TPPFeCl
TPPFeCl
TPPFeCl
TPPFeCl
TPPFeCl
TPPFeCl
TPPFeCl
Aniline
2 min
2 min
2 min
2 min
2 min
2 min
2 min
2 min
97
88
87
66
62
50
67
63
1
2
3
4
5
6
F
F
F
F
S
S
TPPSFeCl
TPPSFeCl
TPPSFeCl
TPPSRuCO
TPPSFeCl
TPPSFeCl
Tryptophan
Phenylalanine
Tyrosine
Tryptophan
Tyrosine
10 min
10 min
10 min
4 heures
10 min
10 min
79
81
51
45
33
51
Tryptophan
Phenylalanine
Tyrosine
Aniline
Tyrosine
a
Tryptophan
Tryptophan
Phenylalanine
Reaction conditions: a solution of substrate/diazo/catalyst mixture (1:1:0.01) in 1 ml
of MeOH/CBS pH (10) (1/1) placed under argon was stirred at 25 C.
a
◦
a
Reaction conditions: a solution of substrate/diazo/(TPPFeCl) mixture (1:1:0.01) in
ml of CH2Cl2 placed under argon at 25 C was stirred for 2 min.
lDiazo and amino ester dissolved in MeOH were added to the buffer solution
◦
1
((CBS pH 10) containing the catalyst and stirred for 4 h).

H.F. Srour et al. / Journal of Molecular Catalysis A: Chemical 407 (2015) 194–203
201
Fig. 4. MALDI-TOF of coupling ethyl diazoacetate (A) and 4-(trifluoromethyl)benzyl diazoacetate (B) to bovine insulin.
Table 4
3
.5. NH insertion of insulin by diazo derivatives in
Insertion of diisopropyl diazomethylphosphonate in N H bond of amino esters
catalyzed by TSPPFeCl in water/methanol.
water-acetonitrile mixture
Substrate
Yield (%)
Insulin is a polypeptide hormone that is critical for the
metabolism of glucose. The insulin monomer consists of two chains,
a 21-residue A chain and a 30-residue B chain, linked by a pair of
1
2
3
4
5
Tryptophan
Tyrosine
Phenylalanine
Aniline
90
92
81
87
96
disulfide bonds, A7 B7 and A20
B 19; an additional intrachain
disulfide bond links A6 and A11. While insulin is synthesized and
stored in the pancreas as a hexamer, the hormonally active form
is the monomer. Because the hormone contains many functional
different groups, it is a good candidate to test our catalytic system
for bioconjugation.
a
Aniline
Reaction conditions: a solution of substrate/diazo/catalyst mixture (1:1:0.01) in 2 ml
of (MeOH:CBS pH 10) (50:50) was stirred at 25 C for 1 h.
◦
a
Solvent: CH2Cl2, catalyst: TPPFeCl.
Coupling was first attempted in the pH range 7.4 using
ethyl diazoacetate and TSPPFeCl as catalyst. Cobaltocene was
used in the insertion of insulin to reduce iron(III) to iron(II).
In the previous experiments, ligation of the amino group of
the amino acid derivatives to iron is supposed to facilitate the
reaction [17]. This is unlikely with insulin. In these experi-
mental conditions, reaction of ethyl diazoacetate with insulin
We previously reported the catalytic effectiveness of ruthenium
porphyrins for carbenoid generation in reactions of diisopropyl dia-
zomethylphosphonate with alkenes (cyclopropanation) [37] and
allylic amine (N H insertion) [38]. We herein report the N H inser-
tion of diazophosphonate into amino acid derivatives catalyzed by
FeTSPPCl (Scheme 4). The results are summarized in Table 4. In
a typical reaction, catalytic insertions into N H bonds were run
in in water/methanol solution at room temperature under argon
atmosphere with a substrate:diazo:catalyst ratio of 100:100:1. As
shown in Table 4, the coupling products are formed with good
yield 81–96% both for aniline and amino acid derivatives without
detection of dimerization. Again, no OH insertion was detected for
tyrosine.
◦
in PBS/acetonitrile (7/4) at 25 c for 1 h afforded the coupling
product with 90% insulin conversion containing 10% of double
insertion (see Fig. 4 and Table 5). Purification was carried out
by dialysis to remove reagents of molecular weight <3000 Da fol-
lowed by HPLC chromatography to remove residual unconjugated
insulin.
Table 5
Alkylation of N-terminus of insulin by TSPPFeCl-catalyzed carbenoid transfer reactions.
Catalyst (conc.)
Diazoa
Conversion (%)
Mono/bisinserted ratio
1
2
3
4
5
6
7
TSPPRuCO (0.17 mM)
TSPPRuCO (0.034 mM)
TSPPFeCl (0.034 mM)
TSPPFeCl (0.034 mM)
TSPPFeCl (0.034 mM)
TSPPFeCl (0.034 mM)
TSPPFeCl (0.17 mM)
EDA (1 mM)
65
14
90
Traces
75
60:5
99:1
90:10
EDA (0.25 mM)
EDA (0.25 mM)
S (0.25 mM)
F (0.25 mM)
DDMP (0.25 mM)
DDMP (0.25 mM)
b
b
b
b
b
98:2
Traces
20
100:0
◦
Reaction conditions: the solution of insulin, diazo, and catalyst was stirred for 1 h at 25 C in a PBS (pH 7.4)/MeCN (7/3) mixture.
a
EDA = ethyl diazoacetate, F = 4-(trifluoromethyl)benzyl diazoacetate and S = 4-(thiomethyl)benzyl diazoacetate, DDMP = diisopropyl diazomethylphosphonate.
one equiv of cobaltocene (vs catalyst) was added.
b

2
02
H.F. Srour et al. / Journal of Molecular Catalysis A: Chemical 407 (2015) 194–203
Fig. 5. Mass spectrum of insulin after ethyl diazoacetate coupling.
Analysis by MALDI-TOF MS upon purification by HPLC, revealed
+
a peak at 5734 + 86 = 5820 (MH ), consistent with the incorporation
of one acetate carbene unit. Electrospray MS/MS (see experimen-
tal section and Fig. 5) of this mono-ligated species as compared to
unmodified insulin, revealed attachment of the ester moiety to the
FVNQHL peptide only, corresponding to the N termini of chain B.
Fragmentation of this peptide yielded peaks at m/z = 206 (ion a ),
1
m/z = 305 (ion a ), m/z = 333 (ion b ), m/z = 447 (ion b ), m/z = 676
2
2
3
(
ion b ) and m/z = 712 (ion b5), according to the modification of the
4
phenylalanine in N-termini. In contrast, fragmentation of chain A,
revealed two unmodified peptides, one from the N-termini (GIVEQ)
and the second to the acid termini (GERGFFYTPKA). This result
showed that neither N termini nor the NH2 of lysine was tethered
by the ester group of the diazo precursor.
In order to confirm this result, 1H NMR analysis of modified
insulin revealed an AB spectrum for the CH2 group, in alpha posi-
tion of NH, located at 3.4 ppm as expected for a chiral amino acid.
In contrast the CH2 group ligated to Gly, revealed a singlet accord-
ing to the results obtained with the amino ester models. Moreover,
the methyl group, of the ligated acetate can also be detected in the
NMR spectrum (Fig. 6).
Fig. 6. 1H NMR of insulin after (A) diazoacetate insertion and, (B) unmodified insulin.
TSPPFeCl was necessary to give 20% conversion with diisopropyl
diazomethylphosphonate.
4. Conclusion
Other diazo derivatives were also tested for coupling to insulin
(
see Table 5). Whereas 4-(methylsulfide)benzyl 2-diazoacetate
This paper suggests that diazo chemistry is not the perfect con-
failed to give any adduct, the coupling of 4-(trifluoromethyl)benzyl
diazoacetate to insulin was quite successful with 75% con-
version and
jugation and assembly technology for all applications, but provides
a powerful, attractive alternative to conventional chemistry. As a
method for applying metal-catalyzed carbenoid transfer reactions
for selective modification of proteins, various amino esters were
a very small amount of bis-insertion (2%) as
detected by MALDI (see Fig. 4B). A stoichiometric amount of

H.F. Srour et al. / Journal of Molecular Catalysis A: Chemical 407 (2015) 194–203
2455–2456.
203
used as models for NH insertion reactions by four different diazo
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with cysteine. The introduction of new functional groups on specific
sites containing NH groups can modify the stability of the macro-
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