Synthetic and mechanistic insight into nosylation of glycine residues

Article abstract of DOI:10.1039/c3ob00014a

The Fukuyama-Mitsunobu alkylation procedure is widely used to introduce alkyl substituents to amino groups in general and N-alkylation of peptides in particular. Here we have investigated the procedure in detail for N-alkylation of peptides with N-termina

Full text of DOI:10.1039/c3ob00014a

Organic &
Biomolecular Chemistry
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Synthetic and mechanistic insight into nosylation of
glycine residues†
Cite this: Org. Biomol. Chem., 2013, 11,
2288
Nicolai Stuhr-Hansen,*a Theis Ivan Søllingb and Kristian Strømgaarda
The Fukuyama–Mitsunobu alkylation procedure is widely used to introduce alkyl substituents to amino
groups in general and N-alkylation of peptides in particular. Here we have investigated the procedure in
detail for N-alkylation of peptides with N-terminal glycine residues, based on the observation that stan-
dard conditions lead to substantial bis-nosylation of the glycine amino group. A systematic evaluation of
this observation was carried out and it was demonstrated that for peptides with alanine, β-alanine or
γ-aminobutyric acid (GABA) as N-terminal residues mono-nosylation was observed under the same con-
ditions. Moreover, bis-nosylation was independent of the type of resin, neighboring amino acid and
nature of the peptide. Calculations suggest that the reason for the bis-nosylation is the fact that the
deprotonated mono-nosyl species is particularly stable in the case of the terminal Gly residue because
the N− residue can become closer to the SO2 unit. Finally, the mono-nosylated N-terminal glycine could
be obtained by careful optimization of the procedure, adding only one equivalent of 2-nitrobenzene-
sulfonyl chloride.
Received 30th July 2012,
Accepted 30th January 2013
DOI: 10.1039/c3ob00014a
www.rsc.org/obc
Introduction
In 1890 Hindsberg1 first reported a N–C coupling method for
smooth alkylation of amines via the corresponding arylsulfon-
amides, thereby avoiding bis-alkylation or even quaternation
of the nitrogen atom (Scheme 1). A highly efficient variant of
the Hindsberg method is the Fukuyama–Mitsunobu method,
which was developed as an alternative utilizing an alcohol as
the alkylating agent.2–4 Here the 2-nitrobenzenesulfonamide is
reacted with alcohol under Mitsunobu conditions, con-
veniently providing the N-alkylated 2-nitrobenzenesulfon-
amide. This procedure has wide applications5 since the
arylsulfonyl group, for ease the 2-nitrobenzenesulfonyl group,
is efficiently removed after alkylation in the presence of thiols
under basic conditions affording solely the mono-alkylated
product (Scheme 1).
Scheme 1
used in the synthesis of peptides and peptide derivatives.7
Particularly N-methylation in peptides is highly efficient.8,9
We have utilized the Fukuyama–Mitsunobu method exten-
sively, both for generating polyamines in the synthesis of
spider polyamine toxins on a solid phase10–15 and, recently, for
N-alkylation of small peptides on a solid phase.16,17 During the
latter studies, we observed that having a glycine residue in the
N-terminal of a small peptide furnished strongly reduced
yields of the desired N-alkyl peptides and realized that this
was due to consistent bis-nosylation of the amino group of
glycine. Here we report a thorough investigation into the
nature of this bis-nosylation. Finally, we also provide a means
to avoid bis-nosylation, which should be a notable precaution
This Fukuyama–Mitsunobu protocol was extended to solid-
phase applications and has been extensively utilized for
N-alkylations on solid phase. Specifically, facile N-alkylation of
peptides using the Fukuyama protocol was demonstrated by
Miller and Scanlan6 and has subsequently been extensively
aDepartment of Drug Design and Pharmacology, University of Copenhagen,
Universitetsparken 2, DK-2100 Copenhagen, Denmark. E-mail: nsh@sund.ku.dk
bDepartment of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100
Copenhagen, Denmark
†Electronic supplementary information (ESI)
available. See DOI:
10.1039/c3ob00014a
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for performing N-alkylation of glycine residues, particularly
considering the widespread use of the Fukuyama–Mitsunobu
protocol.
Results and discussion
As a continuation of investigations aimed at extended appli-
cations of Fukuyama–Mitsunobu solid-phase N-alkylations of
peptides, we introduced 2-nitrobenzenesulfonamide (nosyl)
groups at the N-terminals of dipeptides terminating with
amino acids such as glycine, alanine and valine using a stan-
dard protocol including four equivalents of nosyl chloride,
ensuring almost quantitative sulfonation of amino groups in
analogy to previously reported procedures2 including our own
work.17 In order to check whether the arylsulfonation reactions
had run to completion the product composition from test clea-
vages with TFA was examined by LC-MS. Clean mono-nosyla-
tion was observed in all but one case. Glycine was the notable
exception, since in addition to obtaining the desired mono-
nosylated product, o-Ns-Gly-Gly-OH (1, Scheme 2), LC-MS also
revealed the presence of the corresponding bis-nosylated
product, (o-Ns)2-Gly-Gly-OH (2, Scheme 2). Compounds 1 and
2 were formed in a ratio of approximately 1 : 1 (Scheme 1,
Fig. 1 and Table 1, entry 1), which was verified by subsequent
work-up of the cleavage mixtures affording 1 and 2 in 39% and
42% yields, respectively (Scheme 2).
Fig. 1 Nosylation of resin-bound dipeptides with 4 equivalents of 2-nitro-
benzenesulfonyl chloride followed by cleavage (TFA–TIPS–H2O): (a) Gly–Gly
(Table 1, entry 1); (b) Ala–Gly (Table 1, entry 2); (c) Val–Gly (Table 1, entry 3);
(d) β-Ala–Gly (Table 1, entry 4).
Notably, when alanine or valine residues were present in the
N-terminal and they were treated with nosyl chloride under
identical conditions no bis-nosylation was observed and
exclusively the corresponding mono-nosylated products were
obtained (Fig. 1 and Table 2, entries 2 and 3, respectively). An the C-terminal glycine with valine, but observed that the
obvious rationalization of this phenomenon would be that bis- mono- to bis-nosylation ratio was independent of the neigh-
nosylation was strictly related to the steric bulk at the α-carbon boring amino acid (Table 1, entry 6), suggesting that the steric
of the N-terminal residue. We therefore investigated nosylation influence of the neighboring amino acid had no impact on the
with β-alanine and γ-aminobutyric acid (GABA) in the N-term- nosylation ratio. We then speculated whether bis-nosylation of
inal of a dipeptide under identical conditions. However, in both glycine resides was dependent on the resin and/or linker
cases no bis-nosylation was observed and solely the mono-nosy- employed, and therefore performed the solid-phase nosyla-
lated products could be detected by LC-MS analysis of the TFA tions on a Wang resin instead of on a 2-chlorotrityl resin, and
cleaved reaction mixtures (Fig. 1 and Table 1, entries 4 and 5). obtained similar results (Table 1, entries 7 and 8). Moreover,
This clearly suggests that steric bulk of the α-carbon could not to ensure that the observed bis-nosylation was not restricted to
be a determining factor for the difference between glycine and dipeptides, we prepared two model 15-mer peptides including
alanine or valine in the degree of bis-nosylation.
all proteogenic amino acids bearing side chain functionalities.
In all experiments so far, we employed a dipeptide with When a glycine was the N-terminal residue, the mono- to
glycine in the C-terminal and speculated that the neighboring bis-nosylation ratio was approximately 1 : 1 whereas with an
amino acid could play a decisive role. We therefore replaced N-terminal alanine only mono-nosylation was observed
Scheme 2
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Table 1 N-Terminal mono vs. bis o-nitrobenzenesulfonation of resin bound primary amines
Entry
R
n
Linker
Resin
Mono to bis nosylation ratioa
1
2
3
4
5
6
7
8
H
0
0
0
1
2
0
0
0
0
0
0
0
Gly
Gly
Gly
Gly
Gly
Val
Gly
Val
2-Chlorotrityl
2-Chlorotrityl
2-Chlorotrityl
2-Chlorotrityl
2-Chlorotrityl
2-Chlorotrityl
Wang
50 : 50
100 : 0
100 : 0
100 : 0
100 : 0
50 : 50
50 : 50
50 : 50
50 : 50
100 : 0
100 : 0
50 : 50
Me
Pri
H
H
H
H
H
H
Me
H
Wang
9
YWTSRQPNKHGDCV
YWTSRQPNKHGDCV
–OCH2CH2CvO–
Sarb
2-Chlorotrityl
2-Chlorotrityl
2-Chlorotrityl
2-Chlorotrityl
10
11
12
H
o-Ns = 2-Nitrobenzenesulfonyl (2-O2NC6H4SO2−).a Approximated by HPLC analysis of the cleavage mixture. b Sar (sarcosine) = N-methylglycine.
Table 2 PCM-G3MP2 calculated energies for deprotonationa
Reaction
ΔE
ΔG
1186.7
1190.5
1190.5
1208.1
1194.3
1210.1
1203.7
1202.5
1210.2
1200.4
a 0 K values in kJ mol−1
.
(Table 1, entries 9 and 10), which confirms that this seems to be
a general phenomenon for peptides regardless of their length.
To examine the relevance of these observations to synthesis
in solution (DMF), we performed nosylations using the same
Finally, we turned our attention to the role of the amide conditions applying a large excess of nosyl chloride on tert-
bond between the glycine and the neighboring amino acid. To butylamides of glycine (H-Gly-NHBu-t), alanine (H-Ala-NHBu-t)
address this, we first examined the isosteric replacement of and valine (H-Val-NHBu-t). We observed the same phenom-
the amide functionality with an ester function, thereby remov- enon as on solid phase: For glycine bis-nosylation occurred to
ing the NH moiety and reducing the electronegativity of the a very large degree whereas the amides of alanine and valine
carbonyl group. Performing the nosylation on a Gly–Gly depsi- gave pure mono-nosyl products.
peptide resulted in pure mono-nosylation and the bis-nosy-
Thus, we observed that bis-nosylation occurs for peptides
lated product could not be observed by LC-MS (Table 1, entry with a glycine in the N-terminal. Generally, if a glycine residue
11). This unequivocally demonstrates that the carbonyl group, is nosylated, it will result in a significantly detrimental yield of
the NH moiety or both play a decisive role in the observed bis- the corresponding alkylated peptide. On the other hand, for all
nosylation. To examine this further, and specifically address other investigated residues there seems to be no risk of obtain-
the influence of the amide NH moiety, the N-methylated ing bis-nosylated products when utilizing an excess of nosyl
amino acid, sarcosine (N-methylglycine), was introduced next chloride, as generally employed in solid-phase synthesis. We
to an N-terminal glycine and nosylation was performed. This therefore investigated how the bis-nosylation of glycine resi-
resulted in the same mono- to bis-nosylation ratio of approxi- dues could be avoided using nosylation of the 15 amino acid
mately 1 : 1 (Table 1, entry 12), as observed in our initial peptide GWYTSRQPNKHEDCV on trityl resin as an example.
Gly–Gly dipeptide, thus highlighting the decisive role of the We found that reducing the number of equivalents of nosyl
carbonyl group of the amide bond.
chloride from 4 to 1.1 only led to a very low degree of
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Fig. 2 Nosylation of
a resin-bound 15 amino acid peptide GWYTSRQPN-
KHEDCV with 1.1 equivalents of 2-nitrobenzenesulfonyl chloride leading to an
almost pure mono-nosylated product.
Fig. 3 Calculated geometries (MP2(FULL)/6-31G(d)) for the sulfamide of Gly
and an example of a substituted analogue. The characteristic increase in the
marked dihedral angle upon substitution is noted.
Scheme 3
finding is not in line with the calculated energy for deprotona-
tion in Table 2. We attribute this discrepancy to the fact that
PCM does not take into account specific solvent interactions
and thus the N–H hydrogen bond to the negatively charged
nitrogen will be essential for anion stability and N-methylation
will increase the energy for deprotonation too much compared
to a situation where the solvent can hydrogen bond to the nega-
tively charged nitrogen atom.
bis-nosylation, affording the crude o-Ns-GWYTSRQPNKHEDCV
in high purity (Fig. 2) and a good yield of the mono-nosylated
product (76%) after HPLC purification.
It is important to avoid adding an excess of nosyl chloride
during nosylation of only glycine terminated peptides. Because
of the low amount of bis-nosylated product formed with
addition of 1.1 equivalent (Fig. 2) it is assumed that reaction
with the first nosyl chloride molecule is faster than introduc-
tion of the second nosyl group (Scheme 3).
Conclusions
In conclusion, we identified that bis-nosylation is an impor-
tant issue in treatment of peptides having an N-terminal
glycine with an excess of nosyl chloride. From studies based
on systematic variations of the peptide structure it was clear
that this phenomenon was not caused by steric factors.
Notably, N-terminal glycine exclusively caused bis-nosylation
whereas other amino acid motifs, even with a less hindered
amide functionality, underwent clean mono-nosylation. Intro-
ducing an amide-to-ester substitution next to an N-terminal
glycine cleanly resulted in mono-nosylation whereas an N–Me
group did not, suggesting the carbonyl group of the amide
moiety to be decisive. Calculations indicated that the energy
requirement for deprotonation of the mono-nosylated species
is the key. This energy is determined by the anion stability
which is closely related to the structure as the more substi-
tuted anions are more congested and the N− to S overlap is
thus less easy to form. For use in Fukuyama–Mitsunobu reac-
tions, the formation of bis-nosylated by-products seriously
reduces yield by truncation of the peptide. However, the bis-
nosylation problem could be overcome by using one equivalent
of nosyl chloride since introduction of the first nosyl group is
sufficiently faster than introduction of the second group.
Calculations
In order to understand these observations in more detail, we
employed a computational approach. The 0 K energy was calcu-
lated at the G3(MP2) level which is known to be accurate
within chemical accuracy (2.0 kcal mol−1).18 This method is
based on an MP2(FULL)/6-31G(d) geometry calculation and an
HF/6-31G(d) frequency calculation. We verified that all of the
calculated geometries do correspond to a genuine minimum
(zero imaginary frequencies, Fig. 3). To address solvent effects,
we invoked the polarizable continuum model (PCM) by
Tomasi.19 All calculations were carried out with the
GAUSSIAN03 suite of programs.20
We investigated the central effects in mono- and bis-nosyla-
tion processes computationally to address why a bis-nosylated
product is obtained only in the case of glycine (Table 1) whereas
mono-substitution is obtained for all other amino acids. We
focused on the energy requirements for deprotonation of the
mono-nosylated species as the reaction is carried out under con-
ditions that are sufficiently basic to deprotonate the sulfamide
and because our observations indicate that the bis-nosylation is
related to thermodynamic properties of the process rather than
to kinetics. The results are shown in Table 2 and they are con-
sistent in the sense that the lowest energy of deprotonation is
indeed found for the glycine terminal, and thus it is only in this
case that the mono-nosyl species can become deprotonated to
become sufficiently nucleophilic so as to be attacked by a
Experimental section
Chemistry
second equivalent of NsCl. We observed that bis-nosylation took All reagents were obtained from commercial suppliers and
place when this N–H was methylated (Table 1, entry 12). This used without further purification. Proton (1H) and carbon
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(13C) NMR spectra were recorded on Bruker Avance (400 MHz). while agitating the solution for 1 h followed by draining and
Chemical shifts (δ) are reported in parts per million (ppm) washing with THF, MeOH, and DCM. An inhomogeneous
with reference to tetramethylsilane (TMS) as an internal stan- mixture of TFA–triisopropylsilane (TIPS)–H2O (18 : 1 : 1, 4 mL)
dard. NMR experiments were carried out in CDCl3. The follow- was added and the mixture was shaken for 2 h. The peptide-
ing abbreviations are used for the proton spectra containing solution was filtered into a flask and combined
multiplicities: s, singlet; br s, broad singlet; d, doublet; dd, with two successive washes with DCM. After gentle evaporation
double doublet, triplet; q, quartet; sep, septet m, multiplet. in a stream of nitrogen the crude peptide was analyzed by
Coupling constants (J) are reported in Hertz (Hz). TLC analysis LC-MS and eventually purified by HPLC.
was performed on silica gel F254 (Merck) and detection was
carried out by examination under UV light and staining with
potassium permanganate. Flash column chromatography was
Nosylation of H-Gly-Gly-2ClTrt-resin (Scheme 2 and Table 1,
entry 1)
performed on silica gel with a solvent of HPLC grade. Elemen-
tal analyses were performed by Mr J. Theiner, Department of
Physical Chemistry, University of Vienna, Austria. Preparative
HPLC was performed on an Agilent 1100 system using a C18
reverse-phase column (Zorbax 300 SB-C18, 21.2 × 250 mm)
with a linear gradient of the binary solvent system of H2O–
ACN–TFA (A: 95/5/0.1 and B: 5/95/0.1) and a flow rate of 20 mL
min−1. Analytical HPLC was performed on an Agilent 1100
system with a C18 reverse-phase column (Zorbax 300 SB-C18
column, 4.6 × 150 mm) with a flow rate of 1 mL min−1 and a
linear gradient of the binary solvent system of H2O–ACN–TFA
(A: 95/5/0.1 and B: 5/95/0.1). Mass spectra were obtained with
an Agilent 6410 Triple Quadrupole Mass Spectrometer instru-
ment using electron spray coupled to an Agilent 1200 HPLC
system (ESI-LC/MS) with a C18 reverse-phase column (Zorbax
Eclipse XBD-C18, 4.6 × 50 mm), an auto-sampler and diode-
array detector using a linear gradient of the binary solvent
system of H2O–ACN–formic acid (A: 95/5/0.1 and B: 5/95/0.086)
with a flow rate of 1 mL min−1. During ESI-LC/MS analysis eva-
porative light scattering (ELS) traces were obtained with a
Sedere Sedex 85 Light Scattering Detector. The identity of all
tested compounds was confirmed by ESI-LC/MS, which also
provided purity data (all >95%; UV and ELSD). High-resolution
mass spectra (HRMS) were obtained using a Micromass Q-Tof
2 instrument and were all within 5 ppm of theoretical values.
Solid-phase synthesis nosylated peptides; general procedure.
Peptides were manually synthesized by Fmoc-based solid-
phase peptide synthesis (SPPS) using a MiniBlock (Mettler-
Toledo, Columbus, OH, USA). The 2-chlorotrityl chloride poly-
styrene resin (1–2% DVB cross linking, 100–200 mesh) was
used as a solid support, and after the resin was swelled in dry
DCM for 15–30 min, the first amino acid was loaded to the
resin using diisopropylethylamine (DIPEA) (resin/amino acid/
DIPEA in 1 : 4 : 8) in DCM for 1.5 h, followed by capping with
methanol (DCM–MeOH–DIPEA 17 : 2 : 1). Fmoc deprotection
was performed with 20% piperidine in DMF (1 × 5 and 1 ×
15 min; wash step in between) and coupling of the consecutive
amino acid was carried out with O-(benzotriazol-1-yl)-N,N,N′,
N′-tetramethyluronium hexafluorophosphate (HBTU) and
DIPEA (resin/amino acid/HBTU/DIPEA 1 : 4 : 4 : 4) in dry DMF
(2 mL) for 30 min. After attachment of the last amino acid
Fmoc deprotection was performed, followed by washing and
drying of the resin with DCM. The resin was swelled in DIPEA
(6 equiv.) in THF (2.5 mL) for ca. 30 min. 2-Nitrobenzenesulfo-
nyl (Ns) chloride (4 equiv.) in DCM (1 mL) was added slowly
An approximately 1 : 1 mixture of mono- and bis-nosylated
products was obtained by following the procedure above. Sep-
aration by HPLC gave o-Ns-Gly-Gly-OH 1 and (o-Ns)2-Gly-
Gly-OH 2.
o-Ns-Gly-Gly-OH 1 (Scheme 2)
The mono-nosylated dipeptide 1 (0.031 g, 39%) was obtained
as colorless crystals. Mp: 168–170 °C. Anal. calcd for
C10H11N3O7S: C, 37.86; H, 3.49; N, 13.24. Found: C, 37.67; H,
1
3.23; N, 13.12. H NMR (400 MHz, CDCl3): δ 3.83 (s, 2H), 3.86
(s, 2H), 7.78–7.84 (m, 2H), 7.88–7.92 (m, 1H), 8.07–8.11 (s, 1H).
13C NMR (100 MHz, CDCl3): δ 41.74, 46.60, 126.27, 131.76,
133.83, 134.56, 135.19, 149.45, 170.92, 172.70.
(o-Ns)2-Gly-Gly-OH 2 (Scheme 2)
The bis-nosylated dipeptide 2 (0.053 g, 42%) was obtained as
colorless crystals. Mp: 218–222 °C (dec.). Anal. calcd for
C16H14O11S2: C, 38.25; H, 2.81; N, 11.15. Found: C, 38.39; H,
2.61; N, 11.01. 1H NMR (400 MHz, CDCl3): δ 3.60 (d, J = 5.5 Hz,
2H), 4.54 (s, 2H), 7.89–8.00 (m, 4H), 8.35 (d, J = 8.0 Hz, 2H),
8.43–8.47 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 40.94, 50.72,
124.67, 130.95, 131.80, 132.75, 136.36, 147.42, 165.62, 170.75.
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