Synthesis and Evaluation of Cyclic Acetals of Serine Hydroxylamine for Amide-Forming KAHA Ligations

Article abstract of DOI:10.1055/s-0037-1611635

The α-ketoacid-hydroxylamine (KAHA) ligation allows the coupling of unprotected peptide segments. The most widely used variant employs a 5-membered cyclic hydroxylamine that forms a homoserine ester as the primary ligation product. While very effective, m

Full text of DOI:10.1055/s-0037-1611635

SYNTHESIS
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Georg Thieme Verlag Stuttgart · New York
2019, 51, A–K
paper
A
en
S. Baldauf, J. W. Bode
Paper
Synthesis
Synthesis and Evaluation of Cyclic Acetals of Serine Hydroxyl-
amine for Amide-Forming KAHA Ligations
Simon Baldaufa
Jeffrey W. Bode*a,b
O
O
O
H
N
H
N
O
KAHA ligation
R2
OH
R2
OH
0
0
0
0-0
0
0
1-
8
3
9
4-8
9
1
0
O
X
R1
R1
O
O
α-keto acid
a Laboratorium für Organische Chemie, Department of Chemistry
and Applied Biosciences, ETH Zürich, 8093 Zürich, Switzerland
bode@org.chem.ethz.ch
b Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya
University, Chikusa, Nagoya 464-8602, Japan
cyclic hydroxylamine
serine
Received: 21.11.2018
Accepted: 27.11.2018
Published online: 07.01.2019
DOI: 10.1055/s-0037-1611635; Art ID: ss-2018-z0777-op
a) Our recent work (ref. 5):
O
O
H
H2N
O
R2
N
KAHA ligation
OH
R2
R1
O
O
O
O
License terms:
R1
5-oxaproline
α-keto acid
ester
O
H
N
O
Abstract The α-ketoacid–hydroxylamine (KAHA) ligation allows the
coupling of unprotected peptide segments. The most widely used vari-
ant employs a 5-membered cyclic hydroxylamine that forms a homo-
serine ester as the primary ligation product. While very effective, mono-
mers that give canonical amino acid residues are in high demand. In
order to preserve the stability and reactivity of cyclic hydroxylamines,
but form a canonical amino acid residue upon ligation, we sought to
prepare cyclic derivatives of serine hydroxylamine. An evaluation of sev-
eral cyclization strategies led to cyclobutanone ketals as the leading
structures. The preparation, stability, and amide-forming ligation of
these serine-derived ketals are described.
R2
O to N shift
R1
OH
amide
b) This work:
O
O
O
H
N
H
N
O
KAHA ligation
R2
R2
OH
O
X
R1
R1
O
OH
O
α-keto acid
cyclic hydroxylamine
serine
Scheme 1 KAHA ligation with 5-oxaproline and proposed cyclic serine
hydroxylamine
Key words ligation, hydroxylamines, acetals, amides, peptides
serine residue at the ligation site (Scheme 1). Furthermore,
we describe our efforts to incorporate these novel mono-
mers into a peptide segment.
In 2006, we reported the α-ketoacid–hydroxylamine
(KAHA) amide-forming ligation,1 which complemented the
well-established native chemical ligation2 (NCL) for the
chemical synthesis of proteins. These methods allow che-
moselective couplings of unprotected peptide segments for
the synthesis of peptides and proteins.3 Currently the most
used hydroxylamine for KAHA ligations is 5-oxaproline, due
to its high stability and chemoselective reactivity with α-
ketoacids to form a homoserine residue at the ligation site.4
The primary product of this reaction is an ester, which rear-
ranges to an amide under basic conditions (Scheme 1).5 In
2015, we developed a novel oxazetidine acid which gave
serine at the ligation site, exclusively as the amide product.6
Unfortunately, the synthesis of this monomer is rather long
and it is not stable in its unprotected form.
In designing an alternative, serine-forming ligation
monomer we sought to identify a larger ring structure that
we hoped would be more easily prepared. This consider-
ation led us to consider cyclic variants of L-serine hydroxyl-
amine, including cyclic carbonates, cyclic sulfonates, and
cyclic acetals. To begin, L-serine was converted into the cor-
responding ethyl ester 1. TBS protection of the free alcohol
and subsequent treatment with bromoacetonitrile in the
presence of a base gave 2.
This was treated with a slightly modified protocol from
Fukuyama to give the N-hydroxyl intermediate 3.7 Initially
we envisioned selective Boc-protection of the nitrogen, but
this was unsuccessful under various conditions.8 We in-
stead elected to first protect the free hydroxyl group using
TBSCl followed by N-protection with Fmoc-Cl to give 4. Al-
though initial attempts to remove the TBS protecting group
In this report, we describe the synthesis of a six-mem-
bered cyclic hydroxylamine, which directly yields a native
Georg Thieme Verlag Stuttgart · New York — Synthesis 2019, 51, A–K

B
S. Baldauf, J. W. Bode
Paper
Synthesis
using TBAF led mainly to decomposition, treatment of 4
with concentrated HCl gave clean conversion into diol 5
(Scheme 2).
id 13 to give amide 14 (Table 2). Commonly reported KAHA
conditions use DMSO/H2O at 60 °C, but a HFIP/AcOH mix-
ture showed much better solubility and the reaction was
observed to proceed at 45 °C.11 Therefore all ligation studies
were carried out under these conditions. Almost all mono-
mers tested showed good activity in the KAHA ligation. Af-
ter a period of 12 hours, α-ketoacid 13 was consumed. The
benzylic acetals were stable under the acidic ligation condi-
tions, and electron-poor nitrobenzyl acetal 16 gave slightly
better conversion compared to bromobenzyl acetal 15.
O
O
N
H
N
H2N
HO
1) TBSCl, imidazole, THF
OEt
OEt
2) Bromoacetonitrile, DIPEA, MeCN
TBSO
1
2
O
H
N
1) TBSCl, imidazole, THF
1) m-CPBA, CH2Cl2
HO
OEt
2) Fmoc-Cl, NaHCO3
MeCN/THF/H2O
2) NH2OH•HCl, MeOH
50 °C
TBSO
3
Fmoc
N
O
Fmoc
O
N
HCl, MeOH/THF/H2O
TBSO
OEt
HO
OEt
Table 1 Synthesized Monomers and Their Stabilitya
TBSO
HO
4
5
Monomer
Stability to SPPS cleavage conditions
not stable
Scheme 2 Synthesis of core structure 5
Fmoc
O
N
O
OEt
We explored the formation of the corresponding cyclic
boronic esters, silanes, carbonates, sulfites, and phosphoric
acid esters; however, we were not able to isolate the corre-
sponding six-membered cyclic hydroxylamines. As an alter-
native, we considered cyclic acetals, as similar structures
have proven to be well suited for KAHA ligation.9 For use in
protein synthesis, however, these acid-labile functional
groups would need to survive cleavage from the resin under
standard TFA conditions. Prior to in-depth investigations of
suitable acetals, we carried out preliminary tests to check
that cyclic acetals of 5 could be formed and that Fmoc-re-
moval was feasible. We treated compound 5 with 1,1-dime-
thoxyethylbenzene in the presence of p-toluenesulfonic
acid to give 6 without problem. Removal of the Fmoc group
under basic conditions also proceeded smoothly. With this
promising result we sought to prepare and evaluate various
cyclic acetals.
We initially targeted a methylene acetal, as these are
known to be more resistant to acid cleavage than other
structures.10 Unfortunately, all attempts to form the desired
compound from 5 with various methylene sources using
different catalysts or activation using Brønsted or Lewis ac-
ids did not yield the desired product. Despite this setback,
we continued to synthesize various acetals and ketals by
using either a catalytic amount of p-toluenesulfonic acid for
aldehyde-derived compounds, or In(OTf)3 for the ketone de-
rivatives. Selected acetals and ketals prepared are shown in
Table 1. However, when these compounds were tested for
their stability under SPPS conditions [resin cleavage condi-
tions, TFA/DODT/H2O (95:2.5:2.5 v/v) for 2 h], only cyclobu-
tanone-derivative 12 showed reasonable stability (Table 1).
Despite the poor acid stability, we tested these sub-
strates for activity in KAHA ligation. For this purpose, the
Fmoc protecting group was removed with piperidine and
the monomers were allowed to react with simple α-ketoac-
O
Me
6
Fmoc
O
not stable
not stable
N
O
OEt
O
Br
7
Fmoc
O
N
O
OEt
F3C
O
CF3
8
Fmoc
O
not stable
not stable
N
O
OEt
O
SO2Ph
9
Fmoc
O
N
O
OEt
O
O2N
10
not stable
stable
Fmoc
O
N
O
OEt
EtO
O
11
Fmoc
O
N
O
OEt
O
12
a Stability was tested by treatment with TFA/DODT/H2O (95:2.5:2.5 v/v) for 2 h.
Georg Thieme Verlag Stuttgart · New York — Synthesis 2019, 51, A–K

C
S. Baldauf, J. W. Bode
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Synthesis
Table 2 KAHA Ligation with Selected Monomersa
the presence of Fmoc and the acetal, for the purpose of ap-
plying this monomer in Fmoc-SPPS. Despite all our efforts,
the various conditions only led to total decomposition or
returned starting material without removing the Fmoc pro-
tecting group.
O2N
O
O
O
H
N
H
OH
N
O
R
OEt
OEt
O
O
O2N
O
OH
Monomer
13
14
We instead introduced a benzyl ester at the beginning of
the synthesis. All previously developed steps were compati-
ble with the benzyl-protected starting material, and we
were able to synthesize the benzyl ester analogue 19. Un-
fortunately, deprotection of the benzyl ester using Pd/C un-
der a H2 atmosphere gave mainly decomposition and only
traces of product was observed. We were pleased to see that
exchanging Pd/C for Pd(OH)2 allowed a clean reaction.
Free acid 26 was coupled onto a short peptide segment,
but proved not to be stable upon resin cleavage. Therefore
we prepared and evaluated a number of other ketals from
small rings. Although we were not successful in forming the
product from oxetan-3-one, other cyclobutanone deriva-
tives proved tractable. Brominated derivative 20 was readily
formed and carboxylic esters 21 and 22 could be prepared
from the corresponding acid followed by ester formation.
The stability of compounds 19–22 was tested on the mono-
mer and on a peptide segment (Table 3).
Monomer
Ligation yield after purification
64%
O
H
N
O
OEt
O
Br
15
77%
O
H
N
O
OEt
O
O2N
16
O
no product observed
H
N
O
OEt
EtO
O
17
18
72%
O
H
N
O
OEt
Table 3 Synthesized Monomers and Their Stabilitya
O
Monomer
Stability to SPPS
cleavage conditions conditions on peptide
Stability to SPPS cleavage
monomer 16b
monomer 18c
14
Fmoc
O
stable
stable
not stable
stable
N
O
OBn
14
O
purified
T = 12 h
19
purified
T = 12 h
14
14
Fmoc
O
N
O
OBn
O
13
Br
16
20
13
stable
stable
Fmoc
O
T = 0 h
T = 0 h
N
O
OBn
O
21
MeOOC
a KAHA ligation carried out at 30 mM at 45 °C in HFIP/AcOH with 1% H2O
for 12 h.
stableb
stableb
b HPLC monitoring of the KAHA ligation of α-ketoacid 13 and hydroxyl-
amine 16, and purified amide 14.
Fmoc
O
N
O
OBn
c HPLC monitoring of the KAHA ligation of α-ketoacid 13 and hydroxyl-
amine 18, and purified amide 14.
O
t-BuOOC
22
a Stability was tested by treatment with TFA/DODT/H2O (95:2.5:2.5 v/v) for 2 h.
b Observed as free acid on the cyclobutane ring.
Cyclobutanone-derived acetal 18 was also stable during
the ligation conditions and gave good conversion of the α-
ketoacid. Only 17 did not perform KAHA ligation under
HFIP/AcOH conditions because the deprotected monomer
was not stable under the ligation conditions (Table 2).
This experiment also indicated that open, unsubstituted
serine hydroxylamine does not react under these condi-
tions and that the cyclic structure is essential. With these
promising results, we sought to remove the ethyl ester in
All benzyl-protected monomers were also tested for
their performance in the KAHA ligation. Fmoc removal oc-
curred smoothly in all cases and the deprotected mono-
mers underwent ligation with α-ketoacid 13 in HFIP/AcOH
(Table 4). The cyclobutanone ketal monomers could be con-
verted into the corresponding acid by treatment with
Georg Thieme Verlag Stuttgart · New York — Synthesis 2019, 51, A–K

D
S. Baldauf, J. W. Bode
Paper
Synthesis
Fmoc
O
Pd(OH)2 under H2 atmosphere (Scheme 3) and all com-
pounds tolerated the coupling conditions to introduce the
monomer onto the N-terminus of a peptide segment. Since
OMe ester 21 showed better conversion in the KAHA liga-
tion, we selected this monomer for introduction onto a
short peptide sequence.
Fmoc
O
O
N
O
Pd(OH)2, H2
N
O
OBn
OH
OH
CH2Cl2/EtOH (1:4)
O
O
Fmoc
O
Fmoc
Fmoc
O
N
O
N
O
N
OH
O
OH
O
O
O
MeOOC
Br
26
86%
Table 4 KAHA Ligation with Selected Monomersa
28
90%
27
93%
Fmoc
O
O2N
O
O
O
H
N
N
O
H
OH
OH
N
O
R
OBn
OBn
O
O
O
O2N
O
t-BuOOC
OH
Monomer
13
23
29
96%
Monomer
Ligation yield after purification
79%
Scheme 3 Benzyl deprotection
O
H
N
O
OBn
KAHA ligation performed directly on the unprotected prod-
uct without further purification using α-ketoacid 13 in
HFIP/AcOH. The free hydroxylamine 31 was observed by
LCMS and was completely consumed within 12 hours
(Scheme 4).
O
MeOOC
24
39%
O
H
N
O
OBn
O
Fmoc
O
O
Br
H
N
N
O
25
A Y G NH2
HNEt2
A Y G NH2
O
13 and 24b
13 and 25c
O
O
MeOOC
MeOOC
30
31
23
23
O2N
O
H
N
KAHA ligation with 13
purified
T = 12 h
A Y G NH2
purified
T = 12 h
T = 0 h
O
13
13
OH
23
23
32
13
purified
T = 12 h
T = 0 h
24
25
32
a KAHA ligation carried out at 30 mM at 45 °C in HFIP/AcOH with 1% H2O
for 12 h.
13
b HPLC monitoring of the KAHA ligation of α-ketoacid 13 and hydroxyl-
amine 24, and purified amide 23.
31
T = 0 h
c HPLC monitoring of the KAHA ligation of α-ketoacid 13 and hydroxyl-
amine 25, and purified amide 23.
Scheme 4 Ligation test on a small peptide segment
A short peptide segment with three amino acid resi-
dues, prepared by standard SPPS conditions on a Rink am-
ide resin, was chosen as a model peptide. After deprotection
of the terminal amine, the free acid of monomer 27 was
coupled with HATU/NMM conditions to the resin and
cleaved with TFA/DODT/H2O (95:2.5:2.5 v/v) for 2 hours.
TFA was removed at 40 °C under reduced pressure and the
product was isolated by preparative HPLC. The Fmoc pro-
tecting group was removed with N,N-diethylamine and the
After this positive result, we expanded to a larger pep-
tide fragment consisting of 50 amino acid residues. Al-
though the Fmoc group could be removed cleanly, with a
peak-to-peak conversion by HPLC, the acetal protecting
group unfortunately did not survive the two-step proce-
dure of basic Fmoc-removal conditions and acidic purifica-
tion. The obtained product was 36, the free hydroxylamine
of serine at the N-terminus (Scheme 5).
Georg Thieme Verlag Stuttgart · New York — Synthesis 2019, 51, A–K

E
S. Baldauf, J. W. Bode
Paper
Synthesis
Fmoc O
wavelength UV detector (simultaneous monitoring of the eluent at
220 nm, 254 nm, and 301 nm) and a Rheodyne injector fitted with a
20 or 1000 L injection loop. If required, the columns were heated us-
ing a column heater or a water bath (preparative HPLC). The mobile
phase for RP-HPLC was Milipore-H2O containing 0.1% (v/v) TFA and
HPLC grade MeCN containing 0.1% (v/v) TFA. Analytical HPLC was per-
formed on Shiseido Capcell Pak C18 MGII (5 m, 4.6 mm i.d. × 250
mm) or Shiseido Capcell Pak C18 (UG 80, 5 m, 4.6 mm i.d. × 250 mm)
columns at a flow rate of 1 mL/min. Preparative HPLC was performed
on Shiseido Capcell Pak MGII (5 m, 20 mm i.d. × 250 mm) or Vydac
248MS C18 (10 m, 22 mm i.d. × 250 mm) columns at a flow rate of
10 mL/min.
N
O
OH
VSKVLLSICSLLCDPNPDDPLVPDIAQIYKSDKEKYNRHAREWTQKYANle
O
Br
MeOOC
HOOC
R =
33
34
Conditions
35
O
H
N
OH
HO
VSKVLLSICSLLCDPNPDDPLVPDIAQIYKSDKEKYNRHAREWTQKYANle
36
HO
Scheme 5 Attempts to remove the Fmoc protecting group on a longer
peptide fragment
Ethyl O-(tert-Butyldimethylsilyl)-N-(cyanomethyl)-L-serinate (2)
IR (neat): 2954, 2930, 2857, 1733, 1471, 1251, 1195 cm–1
.
The results were the same regardless of the choice of
base used to remove the Fmoc protecting group, including
basic aqueous solutions. To date, we were not able to over-
come this problem and longer peptides containing the cy-
clic ketal serine hydroxylamines cannot be isolated. A
search for a more stable acetal protecting group is currently
under investigation.
1H NMR (400 MHz, CDCl3):  = 4.19 (q, J = 7.1 Hz, 2 H), 3.92–3.84 (m, 2
H), 3.80–3.59 (m, 2 H), 3.48 (dt, J = 8.2, 4.4 Hz, 1 H), 2.28 (q, J = 7.0 Hz,
1 H), 1.27 (t, J = 7.2 Hz, 3 H), 0.85 (s, 9 H), 0.03 (d, J = 5.7 Hz, 6 H).
13C NMR (101 MHz, CDCl3):  = 171.54 (CO), 117.73 (CN), 64.41 (CH2),
61.61 (CH), 61.39 (CH2), 36.07 (CH2), 25.82 (3 CH3), 18.27 (C), 14.29
(CH3), –5.41 (CH3), –5.55 (CH3).
HRMS (ESI): m/z [M + H]+ calcd for C13H27N2O3Si: 287.1785; found:
In conclusion, we devised a synthetic route to new hy-
droxylamine monomers that yield serine residues upon
KAHA ligation. We found that substituted cyclobutane ke-
tals are stable under acidic conditions and are excellent li-
gation partners. Further investigations are necessary to ap-
ply this monomer on larger peptides, however this strategy
provides a promising new approach to KAHA ligation that
forms canonical amino acid residues at the ligation site.
287.1786.
Ethyl O-(tert-Butyldimethylsilyl)-N-hydroxy-L-serinate (3)
IR (neat): 2953, 2929, 2857, 1737, 1471, 1252, 1107 cm–1
.
1H NMR (400 MHz, CDCl3):  = 6.59 (br s, 1 H), 5.83 (br s, 1 H), 4.21
(qd, J = 7.2, 0.8 Hz, 2 H), 3.88 (qd, J = 10.2, 4.8 Hz, 2 H), 3.74 (dd, J = 5.6,
4.0 Hz, 1 H), 1.27 (t, J = 7.1 Hz, 3 H), 0.85 (s, 9 H), 0.03 (d, J = 3.3 Hz, 6
H).
13C NMR (101 MHz, CDCl3):  = 171.21 (CO), 66.83 (CH), 61.21 (CH2),
61.08 (CH2), 25.85 (3 CH3), 18.29 (C), 14.31 (CH3), –5.46 (CH3), –5.53
(CH3).
Fmoc-amino acids with suitable side-chain protecting groups, HATU
(1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridini-
um 3-oxide hexafluorophosphate) were purchased from Peptides In-
ternational (Louisville, KY, USA) and ChemImpex (Wood Dale, IL,
USA). Solvents for flash chromatography (EtOAc, hexanes, MeOH)
were of technical grade and distilled prior to use. HPLC grade MeCN
from Sigma-Aldrich was used for analytical and preparative HPLC pu-
rification. DMF (>99.8%) from Sigma-Aldrich was directly used with-
out further purification for solid phase peptide synthesis. Other com-
mercially available reagents and solvents were purchased from Sig-
ma-Aldrich (Buchs, Switzerland), Acros Organics (Geel, Belgium), and
HRMS (ESI): m/z [M + H]+ calcd for C11H26NO4Si: 264.1626; found:
264.1627.
Ethyl N-(tert-Butyldimethylsiloxy)-O-(tert-butyldimethylsilyl)-N-
[(9H-fluoren-9-ylmethoxy)carbonyl]-L-serinate (4)
IR (neat): 2953, 2928, 2856, 1738, 1463, 1249, 1095 cm–1
.
1H NMR (400 MHz, CDCl3):  = 7.79 (dt, J = 7.6, 0.9 Hz, 2 H), 7.69 (dt,
J = 7.5, 1.0 Hz, 2 H), 7.42 (tt, J = 7.5, 0.8 Hz, 2 H), 7.32 (tt, J = 7.4, 1.1 Hz,
2 H), 4.72 (dd, J = 7.9, 6.3 Hz, 1 H), 4.55–4.39 (m, 2 H), 4.31 (t, J = 7.1
Hz, 1 H), 4.21 (qd, J = 7.1, 3.2 Hz, 2 H), 4.10 (dd, J = 7.2, 1.8 Hz, 2 H),
1.28 (t, J = 7.1 Hz, 3 H), 0.96 (s, 9 H), 0.91 (s, 9 H), 0.21 (d, J = 22.0 Hz, 6
H), 0.09 (s, 6 H).
13C NMR (101 MHz, CDCl3):  = 168.68 (CO), 159.38 (CO), 143.89 (C),
143.82 (C), 141.44 (C), 141.42 (C), 127.87 (CH), 127.86 (CH), 127.21 (2
CH), 125.49 (CH), 125.43 (CH), 120.10 (CH), 120.09 (CH), 68.36 (CH2),
66.93 (CH), 61.45 (CH2), 59.85 (CH2), 47.13 (CH), 26.09 (3 CH3), 26.00
(3 CH3), 18.41 (2 C), 14.32 (CH3), –4.70 (CH3), –4.73 (CH3), –5.21 (CH3),
–5.29 (CH3).
TCI Europe (Zwijndrecht, Belgium). DODT
= 2,2′-(ethylenedi-
1
oxy)diethanethiol. H and 13C NMR spectra were recorded on Bruker
DRX400, Bruker AVIII400 and Bruker AVIII600 spectrometers. HRMS
were recorded by the Mass Service of the Laboratory of Organic
Chemistry at ETH Zurich either with a Bruker maXis instrument (ESI-
MS measurements) equipped with an ESI source and a Qq-TOF detec-
tor or with a Bruker solariX instrument (MALDI-FTICR-MS) using 4-
hydroxy--cyanocinnamic acid as matrix. All reactions were per-
formed using standard techniques under an atmosphere of N2. Reac-
tions and fractions from flash chromatography were monitored by
TLC using aluminum plates (Merck, TLC Silica gel 60 W F254s) and vi-
sualized by staining with basic KMnO4 solution or acidic ninhydrin
solution. Flash chromatography was performed on Silicycle SiO2 Type
F60 (230–400 mesh) using a forced flow of air at 0.5–1.0 bar. Unless
otherwise stated, peptides and protein segments were analyzed and
purified by RP-HPLC on Jasco analytical and preparative instruments
equipped with dual pumps, mixer and in-line degasser, a variable
HRMS (ESI): m/z [M + H]+ calcd for C32H50NO6Si2: 600.3171; found:
600.3174.
Ethyl N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N-hydroxy-L-seri-
nate (5)
IR (neat): 2956, 1730, 1700, 1450, 1312, 1111, 1047 cm–1
.
Georg Thieme Verlag Stuttgart · New York — Synthesis 2019, 51, A–K

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S. Baldauf, J. W. Bode
Paper
Synthesis
1H NMR (400 MHz, CDCl3):  = 7.78 (dt, J = 7.6, 1.0 Hz, 2 H), 7.62 (t, J =
6.2 Hz, 2 H), 7.42 (tt, J = 7.5, 1.3 Hz, 2 H), 7.36–7.29 (m, 2 H), 4.86 (s, 1
H), 4.57–4.42 (m, 2 H), 4.35–4.21 (m, 3 H), 4.21–4.06 (m, 3 H), 2.74 (s,
1 H), 1.29 (td, J = 7.1, 5.9 Hz, 3 H).
Benzyl N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N-hydroxy-L-seri-
nate (benzyl-5)
IR (neat): 3674, 3305, 2969, 2900, 1738, 1708, 1450, 1262, 1106, 1048
cm–1
.
13C NMR (101 MHz, CDCl3):  = 158.33 (CO), 143.54 (2 C)*, 143.48 (2
C), 141.43 (2 C)*, 141.43 (2 C), 128.02 (2 CH)*, 128.02 (2 CH), 127.32
(2 CH)*, 127.30 (2 CH), 125.25 (2 CH)*, 125.20 (2 CH), 120.19 (2 CH),
69.06 (CH2), 64.07 (CH), 62.29 (CH2), 60.57 (CH2), 47.02 (CH), 14.35
(CH3)*, 14.26 (CH3); * signals of rotamers.
HRMS (MALDI): m/z [M + Na]+ calcd for C20H21NO6Na: 394.1261;
found: 394.1261.
1H NMR (400 MHz, CDCl3):  = 7.73 (ddt, J = 7.6, 2.1, 0.9 Hz, 2 H), 7.53
(t, J = 8.3 Hz, 2 H), 7.37 (tdt, J = 7.5, 3.3, 0.8 Hz, 2 H), 7.35–7.17 (m, 7
H), 5.18 (s, 2 H), 4.88 (br s, 1 H), 4.47–4.34 (m, 2 H), 4.22–4.07 (m, 3
H), 3.13 (br s, 1 H); 1 H not observed.
13C NMR (101 MHz, CDCl3):  = 168.65 (CO), 158.95 (CO), 143.39 (2 C),
141.37 (2 C), 134.97 (C), 128.76 (2 CH), 128.65 (CH), 128.22 (2 CH),
127.99 (2 CH)*, 127.97 (2 CH), 127.31 (2 CH)*, 127.29 (2 CH), 125.21
(2 CH)*, 125.16 (2 CH), 120.14 (2 CH), 69.16 (CH2), 67.70 (CH2), 64.31
(CH), 59.96 (CH2), 46.91 (CH); * signals of rotamers.
Benzyl O-(tert-Butyldimethylsilyl)-N-(cyanomethyl)-L-serinate
(benzyl-2)
HRMS (ESI): m/z [M + Na]+ calcd for C25H23NNaO6: 456.1418; found:
456.1419.
IR (neat): 3674, 2987, 2956, 1736, 1463, 1406, 1252, 1083 cm–1
.
1H NMR (400 MHz, CDCl3):  = 7.40–7.31 (m, 5 H), 5.19 (s, 2 H), 3.97–
3.87 (m, 2 H), 3.81–3.71 (m, 1 H), 3.71–3.53 (m, 2 H), 2.31 (br s, 1 H),
0.86 (s, 9 H), 0.02 (d, J = 10.2 Hz, 6 H).
13C NMR (101 MHz, CDCl3):  = 171.48 (CO), 135.39 (C), 128.76 (2 CH),
128.60 (CH), 128.49 (2 CH), 117.71 (CN), 67.17 (CH2), 64.37 (CH2),
61.68 (CH), 36.07 (CH2), 25.85 (3 CH3), 18.29 (C), –5.40 (CH3), –5.51
(CH3).
Ketal Formation with In(OTf)3; General Procedure
In(OTf)3 (0.30 equiv) was added to a solution of 5 (1.00 equiv) and ke-
tone (5.00 equiv) in CH2Cl2 (0.15 M) at 0 °C. The reaction was allowed
to warm up to r.t. and was stirred overnight. The solution was diluted
with CH2Cl2 and 10% citric acid solution was added. The phases were
separated and the aqueous layer was extracted with CH2Cl2 (2 ×). The
combined organic layers were washed with sat. aq NaHCO3 solution
and brine and dried (Na2SO4). The drying agent was removed by fil-
tration and the solvent was evaporated. The residue was purified by
flash chromatography.
HRMS (ESI): m/z [M + Na]+ calcd for C18H28N2NaO3Si: 371.1761 found
371.1761.
Benzyl O-(tert-Butyldimethylsilyl)-N-hydroxy-L-serinate (benzyl-
3)
3-Ethyl 2-(9H-Fluoren-9-ylmethyl) (3S)-6-Methyl-6-phenyl-1,5,2-
dioxazinane-2,3-dicarboxylate (6)
IR (neat): 3674, 2987, 2900, 1739, 1462, 1252, 1192, 1105, 1066 cm–1
.
1H NMR (400 MHz, CDCl3):  = 7.40–7.29 (m, 5 H), 5.87 (br s, 1 H),
5.58 (br s, 1 H), 5.21 (s, 2 H), 3.97–3.87 (m, 2 H), 3.81 (dd, J = 5.2, 4.1
Hz, 1 H), 0.86 (s, 9 H), 0.03 (s, 3 H), 0.02 (s, 3 H).
13C NMR (101 MHz, CDCl3):  = 171.19 (CO), 135.58 (C), 128.74 (2 CH),
128.52 (CH), 128.41 (2 CH), 66.97 (CH2), 66.90 (CH), 61.18 (CH2),
25.89 (3 CH3), 18.33 (C), –5.43 (CH3), –5.48 (CH3).
IR (neat): 2987, 2900, 1713, 1449, 1304, 1098, 1065 cm–1
.
1H NMR (500 MHz, CDCl3):  = 7.78 (ddt, J = 7.6, 5.7, 0.9 Hz, 2 H), 7.77–
7.62 (m, 2 H), 7.56–7.52 (m, 2 H), 7.46–7.27 (m, 7 H), 4.76–4.65 (m, 2
H), 4.45 (dd, J = 10.5, 7.7 Hz, 1 H), 4.38 (dd, J = 3.9, 1.6 Hz, 2 H), 4.33 (t,
J = 7.1 Hz, 1 H), 4.20 (qd, J = 7.1, 1.6 Hz, 2 H), 1.78 (s, 3 H), 1.20 (t, J =
7.1 Hz, 3 H).
13C NMR (126 MHz, CDCl3):  = 168.07 (CO), 156.52 (CO), 144.05 (C),
143.43 (C), 141.57 (C), 141.46 (C), 140.67 (C), 128.82 (CH), 128.30 (2
CH), 127.97 (2 CH)*, 127.92 (2 CH), 127.25 (2 CH)*, 127.20 (2 CH),
125.66 (CH), 125.35 (2 CH), 125.24 (CH), 120.14 (2 CH)*, 120.10 (2
CH), 103.89 (C), 68.23 (CH2), 62.16 (CH2), 59.91 (CH2), 57.53 (CH),
47.14 (CH), 21.58 (CH3), 14.22 (CH3); * signals of rotamers/diastereo-
mers.
HRMS (ESI): m/z [M + H]+ calcd for C16H28NO4Si: 326.1782; found:
326.1779.
Benzyl N-(tert-Butyldimethylsiloxy)-O-(tert-butyldimethylsilyl)-
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-serinate (benzyl-4)
IR (neat): 3674, 2956, 1740, 1450, 1361, 1250, 1098, 1076, 1005 cm–1
1H NMR (400 MHz, CDCl3):  = 7.75 (dq, J = 7.5, 0.9 Hz, 2 H), 7.63–7.59
(m, 2 H), 7.46–7.18 (m, 9 H), 5.23–5.10 (m, 2 H), 4.71 (dd, J = 7.6, 6.5
Hz, 1 H), 4.49–4.35 (m, 2 H), 4.19 (t, J = 7.0 Hz, 1 H), 4.14–4.07 (m, 2
H), 0.93 (s, 9 H), 0.88 (s, 9 H), 0.20 (s, 3 H), 0.15 (s, 3 H), 0.07 (s, 3 H),
0.06 (s, 3 H).
.
HRMS (MALDI): m/z [M + H]+ calcd for C28H28NO6: 474.1911; found:
474.1907.
7-Ethyl 6-(9H-Fluoren-9-ylmethyl) (S)-5,9-Dioxa-6-
13C NMR (101 MHz, CDCl3):  = 168.55 (CO), 159.37 (CO), 143.85 (2
C)*, 143.78 (2 C), 141.41 (2 C)*, 141.38 (2 C), 135.47 (C), 128.62 (2
CH), 128.36 (CH), 128.27 (2 CH), 127.82 (2 CH)*, 127.80 (2 CH), 127.19
(2 CH), 125.40 (2 CH)*, 125.35 (2 CH), 120.06 (2 CH)*, 120.04 (2 CH),
68.29 (CH2), 67.16 (CH), 67.08 (CH2), 59.96 (CH2), 47.08 (CH), 26.08 (3
CH3), 25.99 (3 CH3), 18.39 (C), 18.37 (C), –4.74 (2 CH3), –5.23 (CH3),
–5.30 (CH3); * signals of rotamers.
azaspiro[3.5]nonane-6,7-dicarboxylate (12)
IR (neat): 3674, 2987, 2901, 1714, 1450, 1408, 1313, 1058 cm–1
.
1H NMR (500 MHz, CDCl3):  = 7.83–7.78 (m, 2 H), 7.78–7.62 (m, 2 H),
7.48–7.41 (m, 2 H), 7.38–7.31 (m, 2 H), 4.66 (dd, J = 10.3, 6.6 Hz, 1 H),
4.55 (s, 1 H), 4.43 (t, J = 9.2 Hz, 1 H), 4.40–4.33 (m, 2 H), 4.30 (q, J = 7.1
Hz, 2 H), 4.05 (dd, J = 11.5, 3.8 Hz, 1 H), 2.49–2.36 (m, 2 H), 2.36–2.24
(m, 2 H), 1.99–1.80 (m, 2 H), 1.29 (t, J = 7.1 Hz, 3 H).
13C NMR (126 MHz, CDCl3):  = 168.06 (CO), 156.43 (CO), 144.05 (2
C)*, 143.37 (2 C), 141.50 (2 C)*, 141.43 (2 C), 127.98 (2 CH)*, 127.93 (2
CH), 127.25 (2 CH)*, 127.12 (2 CH), 125.58 (2 CH)*, 125.18 (2 CH),
120.15 (2 CH)*, 120.13 (2 CH), 104.99 (C), 68.50 (CH2), 62.34 (CH2),
60.27 (CH2), 57.22 (CH), 47.03 (CH), 32.70 (CH2), 30.37 (CH2), 14.23
(CH3), 11.59 (CH2); * signals of rotamers/diastereomers.
HRMS (ESI): m/z [M + H]+ calcd for C37H52NO6Si2: 662.3328; found:
662.3323.
Georg Thieme Verlag Stuttgart · New York — Synthesis 2019, 51, A–K

G
S. Baldauf, J. W. Bode
Paper
Synthesis
HRMS (ESI): m/z [M + Na]+ calcd for C24H25NNaO6: 446.1574; found:
HRMS (ESI): m/z [M + H]+ calcd for C30H28NO8: 530.1809; found:
446.1575.
530.1808.
7-Benzyl 6-(9H-Fluoren-9-ylmethyl) (S)-5,9-Dioxa-6-
7-Benzyl 6-(9H-Fluoren-9-ylmethyl) 2-Methyl (S)-5,9-Dioxa-6-
azaspiro[3.5]nonane-6,7-dicarboxylate (19)
azaspiro[3.5]nonane-2,6,7-tricarboxylate (21)
IR (neat): 3674, 2987, 2900, 1746, 1716, 1451, 1407, 1260, 1098, 1065
IR (neat): 3674, 2987, 2900, 1729, 1450, 1406, 1285, 1090, 1064 cm–1
.
cm–1
.
1H NMR (400 MHz, CDCl3):  = 7.80–7.75 (m, 2 H), 7.62 (ddd, J = 41.3,
13.0, 8.3 Hz, 2 H), 7.46–7.20 (m, 9 H), 5.24 (d, J = 3.1 Hz, 2 H), 4.65–
4.34 (m, 3 H), 4.36–4.15 (m, 2 H), 4.09–3.94 (m, 1 H), 3.71 (d, J = 23.0
Hz, 3 H), 3.11–2.94 (m, 1 H), 2.85–2.40 (m, 4 H).
1H NMR (500 MHz, CDCl3):  = 7.77 (dq, J = 7.6, 1.1 Hz, 2 H), 7.75–7.54
(m, 2 H), 7.47–7.21 (m, 9 H), 5.25 (s, 2 H), 4.56 (dd, J = 10.4, 6.7 Hz, 2
H), 4.40–4.31 (m, 2 H), 4.22 (t, J = 7.4 Hz, 1 H), 4.17–4.00 (m, 1 H),
2.50–2.19 (m, 4 H), 1.99–1.78 (m, 2 H).
13C NMR (126 MHz, CDCl3):  = 168.05 (CO), 156.45 (CO), 144.47 (2
C)*, 144.05 (2 C)*, 143.34 (2 C), 141.66 (2 C)*, 141.49 (2 C*), 141.41 (2
C), 135.22 (C), 128.63 (2 CH), 128.44 (CH), 128.09 (2 CH), 127.97 (2
CH)*, 127.91 (2 CH)*, 127.73 (2 CH), 127.24 (2 CH)*, 127.11 (2 CH),
125.60 (2 CH)*, 125.18 (2 CH), 124.84 (2 CH), 120.20 (2 CH)*, 120.13
(2 CH)*, 120.10 (2 CH), 105.02 (C), 68.47 (CH2), 67.87 (CH2), 60.30
(CH2), 57.34 (CH), 46.96 (CH), 32.72 (CH2), 30.41 (CH2), 11.60 (CH2); *
signals of rotamers/diastereomers.
13C NMR (101 MHz, CDCl3):  = 174.67 (CO)*, 174.35 (CO), 171.26
(CO), 167.85 (CO)*, 167.75 (CO), 143.93 (2 C)*, 143.37 (2 C)*, 143.25 (2
C), 141.50 (2 C)*, 141.43 (2 )*, 141.33 (2 C), 135.11 (C), 128.65 (2 CH)*,
128.63 (2 CH), 128.50 (CH)*, 128.48 (CH), 128.12 (2 CH), 128.01 (2
CH)*, 127.97 (2 CH)*, 127.93 (2 CH), 127.36 (2 CH)*, 127.28 (2 CH)*,
127.26 (2 CH)*, 127.15 (2 CH), 125.72 (2 CH)*, 125.37 (2 CH)*, 125.05
(2 CH), 120.17 (2 CH)*, 120.14 (2 CH)*, 120.08 (2 CH)*, 120.05 (2 CH),
101.79 (C), 101.57 (C), 68.90 (CH2)*, 68.36 (CH2), 67.98 (CH2)*, 67.95
(CH2), 60.63 (CH2)*, 60.53 (CH2), 57.57 (CH)*, 57.37 (CH), 52.26 (CH3),
46.99 (CH)*, 46.84 (CH), 36.57 (CH2)*, 36.42 (CH2), 34.73 (CH2)*, 34.28
(CH2), 28.85 (C), 28.67 (CH); * signals of rotamers/diastereomers.
HRMS (ESI): m/z [M + NH4]+ calcd for C29H31N2O6: 503.2177; found:
503.2174.
HRMS (MALDI/ESI): m/z [M + Na]+ calcd for C31H29NNaO8: 566.1785;
found: 566.1786.
7-Benzyl 6-(9H-Fluoren-9-ylmethyl) (S)-2-Bromo-5,9-dioxa-6-
azaspiro[3.5]nonane-6,7-dicarboxylate (20)
7-Benzyl 2-tert-Butyl 6-(9H-Fluoren-9-ylmethyl) (S)-5,9-Dioxa-6-
azaspiro[3.5]nonane-2,6,7-tricarboxylate (22)
IR (neat): 3674, 2987, 2900, 1732, 1720, 1406, 1393, 1275, 1065, 1056
cm–1
.
IR (neat): 3674, 2974, 2900, 1722, 1451, 1366, 1283, 1157, 1089, 1012
1H NMR (400 MHz, CDCl3):  = 7.76 (ddd, J = 8.4, 2.7, 1.0 Hz, 2 H), 7.55
(dd, J = 33.2, 7.5 Hz, 2 H), 7.43–7.22 (m, 9 H), 5.19 (s, 2 H), 4.62–4.32
(m, 4 H), 4.24–4.04 (m, 2 H), 3.90 (dd, J = 11.6, 3.7 Hz, 1 H), 3.03–2.87
(m, 2 H), 2.70–2.46 (m, 2 H).
13C NMR (101 MHz, CDCl3):  = 167.56 (CO), 156.01 (CO), 143.81 (2
C)*, 143.15 (2 C), 141.53 (2 C)*, 141.44 (2 C), 135.04 (C), 128.65 (2
CH), 128.52 (CH), 128.12 (2 CH), 128.03 (2 CH)*, 128.01 (2 CH), 127.26
(2 CH)*, 127.19 (2 CH), 125.19 (2 CH)*, 124.94 (2 CH), 120.20 (2 CH)*,
120.12 (2 CH), 102.37 (C), 67.97 (CH2), 65.98 (CH2), 60.86 (CH2), 57.26
(CH), 47.07 (CH), 45.50 (CH2), 43.97 (CH2), 31.60 (CH); * signals of ro-
tamers/diastereomers.
cm–1
.
1H NMR (400 MHz, MeOD):  = 7.79 (ddt, J = 7.6, 3.1, 0.9 Hz, 2 H), 7.61
(dt, J = 27.2, 7.6 Hz, 2 H), 7.51–7.14 (m, 9 H), 5.39–5.02 (m, 3 H), 4.67–
3.89 (m, 5 H), 2.98–2.14 (m, 5 H), 1.51–1.40 (m, 9 H).
13C NMR (101 MHz, MeOD):  = 175.06 (CO)*, 174.93 (CO), 169.28
(CO)*, 169.17 (CO), 157.46 (CO), 145.13 (2 C)*, 144.99 (2 C)*, 144.70 (2
C), 142.69 (2 C)*, 142.63 (2 C)*, 142.56 (2 C)*, 142.51 (2 C), 136.75
(C)*, 136.69 (C), 129.53 (2 CH)*, 129.49 (2 CH)*, 129.47 (2 CH), 129.28
(CH), 129.08 (2 CH)*, 129.03 (2 CH)*, 128.96 (2 CH), 128.91 (2 CH)*,
128.90 (2 CH), 128.31 (2 CH)*, 128.26 (2 CH)*, 128.22 (2 CH)*, 128.17
(2 CH), 126.52 (2 CH)*, 126.29 (2 CH)*, 126.15 (2 CH)*, 125.96 (2 CH),
120.98 (2 CH)*, 120.94 (2 CH), 102.82 (C)*, 102.75 (C), 81.99 (C)*,
81.93 (C), 69.71 (CH), 69.02 (CH2)*, 68.62 (CH2), 68.56 (CH2)*, 67.91
(CH2), 61.73 (CH2)*, 61.44 (CH2), 59.36 (CH)*, 58.99 (CH), 48.22 (CH)*,
47.98 (CH), 37.03 (CH2)*, 36.86 (CH2), 35.34 (CH2)*, 34.85 (CH2), 31.19
(CH)*, 30.93 (CH)*, 30.59 (CH), 28.29 (3 CH3)*, 28.25 (3 CH3); * signals
of rotamers/diastereomers.
HRMS (MALDI/ESI): m/z [M
+
Na]+ calcd for C29H26BrNNaO6:
586.0836; found: 586.0835.
(S)-7-(Benzyloxycarbonyl)-6-[(9H-fluoren-9-ylmethoxy)carbon-
yl]-5,9-dioxa-6-azaspiro[3.5]nonane-2-carboxylic Acid (21a)
IR (neat): 3674, 2987, 2900, 1736, 1450, 1393, 1264, 1105, 1065 cm–1
.
1H NMR (400 MHz, CDCl3):  = 10.67 (br s, 1 H), 7.81–7.73 (m, 2 H),
7.72–7.52 (m, 2 H), 7.48–7.21 (m, 9 H), 5.25 (s, 2 H), 4.72–4.15 (m, 5
H), 4.11–3.91 (m, 1 H), 3.09–2.95 (m, 1 H), 2.84–2.38 (m, 4 H).
HRMS (MALDI/ESI): m/z [M + Na]+ calcd for C34H35NNaO8: 608.2255;
found: 608.2257.
13C NMR (101 MHz, CDCl3):  = 179.80 (CO)*, 179.74 (CO), 167.74
(CO)*, 167.67 (CO), 156.57 (CO)*, 156.20 (CO), 143.80 (2 C)*, 143.74 (2
C)*, 143.32 (2 C)*, 143.14 (2 C), 141.43 (2 C)*, 141.34 (2 C)*, 141.29 (2
C)*, 141.22 (2 C), 135.03 (C)*, 134.99 (C), 128.57 (2 CH)*, 128.55 (2
CH), 128.42 (CH)*, 128.40 (CH), 128.02 (2 CH)*, 128.01 (2 CH), 127.95
(2 CH)*, 127.93 (2 CH)*, 127.87 (2 CH)*, 127.83 (2 CH), 127.24 (2 CH)*,
127.19 (2 CH)*, 127.10 (2 CH), 125.52 (2 CH)*, 125.31 (2 CH)*, 125.26
(2 CH)*, 124.95 (2 CH), 120.10 (2 CH)*, 120.08 (2 CH)*, 119.96 (2 CH),
101.56 (C), 68.25 (CH2)*, 67.90 (CH2)*, 67.87 (CH2), 60.53 (CH2)*, 60.42
(CH2), 57.44 (CH), 46.91 (CH)*, 46.72 (CH), 36.34 (CH2)*, 36.15 (CH2),
34.65 (CH2)*, 34.21 (CH2), 28.84 (C), 28.71 (CH); * signals of rotam-
ers/diastereomers.
6-[(9H-Fluoren-9-yl)methyl] 2-Methyl (S)-7-{(S)-3-[2-{(S)-1-[(2-
Amino-2-oxoethyl)amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-
yl}hydrazineyl]-2-oxobutanoyl}-5,9-dioxa-6-azaspiro[3.5]nonane-
2,6-dicarboxylate (30)
HRMS (ESI): m/z [M + Na]+ calcd for C38H41N5NaO11: 766.2695; found:
766.2696.
HPLC (Shiseido Capcell Pak UG80 C18 column (4.6 × 250 mm), heated
to 60 °C, 10 to 90% CH3CN with 0.1% TFA in 20 min): tR = 24.8 min.
Segment 33
HRMS (ESI): m/z [M]+ calcd for C282H431BrN70O83S2: 6269.0282; found:
6269.0453.
Georg Thieme Verlag Stuttgart · New York — Synthesis 2019, 51, A–K

H
S. Baldauf, J. W. Bode
Paper
Synthesis
HPLC (Shiseido Capcell Pak UG80 C18 column (4.6 × 250 mm), heated
3-Ethyl 2-(9H-Fluoren-9-ylmethyl) (3S)-6-[(Phenylsulfonyl)meth-
to 60 °C, 10 to 90% CH3CN with 0.1% TFA in 20 min): tR = 24.9 min.
yl]-1,5,2-dioxazinane-2,3-dicarboxylate (9)
IR (neat): 2987, 1736, 1447, 1306, 1147, 1116, 1065 cm–1
.
Segment 34
1H NMR (500 MHz, CDCl3):  = 8.11–7.24 (m, 13 H), 5.05 (ddd, J = 20.8,
6.6, 4.1 Hz, 1 H), 4.50 (ddd, J = 14.1, 10.4, 7.3 Hz, 1 H), 4.45–4.29 (m, 2
H), 4.28–4.19 (m, 2 H), 4.17–4.00 (m, 0.5 H), 3.95 (dd, J = 10.1, 4.2 Hz,
0.5 H), 3.54 (dt, J = 14.5, 7.4 Hz, 1 H), 3.39 (dt, J = 14.3, 4.2 Hz, 1 H),
3.31 (d, J = 6.1 Hz, 2 H), 1.27 (q, J = 7.1 Hz, 3 H).
HRMS (ESI): m/z [M]+ calcd for C284H434N70O85S2: 6249.1231; found:
6249.0065.
HPLC (Shiseido Capcell Pak UG80 C18 column (4.6 × 250 mm), heated
to 60 °C, 10 to 90% CH3CN with 0.1% TFA in 20 min): tR = 24.5 min.
13C NMR (126 MHz, CDCl3):  = 168.26 (CO), 157.70 (CO), 143.93 (C),
143.70 (C)*, 141.42 (C), 141.36 (C), 139.67 (C), 139.48 (C), 134.18
(CH), 129.47 (2 CH), 128.15 (2 CH), 128.03 (CH), 127.89 (CH), 127.26
(2 CH), 125.62 (CH), 125.42 (CH), 120.08 (2 CH), 98.92 (CH)*, 97.72
(CH), 68.79 (CH2)*, 68.72 (CH2), 62.22 (CH2)*, 62.19 (CH2)*, 61.94
(CH2), 59.35 (CH2)*, 58.27 (CH2), 53.83 (CH2), 47.05 (CH), 14.27 (CH3);
* signals of rotamers/diastereomers.
Segment 35
HRMS (ESI): m/z [M]+ calcd for C284H433N69O85S2: 6234.1122; found:
6235.1309.
HPLC (Shiseido Capcell Pak UG80 C18 column (4.6 × 250 mm), heated
to 60 °C, 10 to 90% CH3CN with 0.1% TFA in 20 min): tR = 24.1 min.
HRMS (ESI): m/z [M + H]+ calcd for C28H28NO8S: 538.1530; found:
538.1524.
Acetal Formation with p-Toluenesulfonic Acid Monohydrate; Gen-
eral Procedure
Dimethyl acetal (2.00 equiv) and p-toluenesulfonic acid monohydrate
(0.10 equiv) were added to 5 (1.00 equiv) in DMF (0.05 M). The solu-
tion was stirred at 50 °C under vacuum for 4 h. The resulting dark
brown viscous gel was diluted with CH2Cl2 and sat. aq NaHCO3 solu-
tion was added to the solution. The aqueous layer was extracted with
CH2Cl2 (2 ×). The combined organic layers were washed with brine
and dried (MgSO4). The drying agent was removed by filtration and
the solvent was evaporated. The residue was purified by flash chro-
matography.
3-Ethyl 2-(9H-Fluoren-9-ylmethyl) (3S)-6-(4-Nitrophenyl)-1,5,2-
dioxazinane-2,3-dicarboxylate (10)
IR (neat): 3674, 2987, 2901, 1724, 1524, 1450, 1349, 1242, 1080 cm–1
.
1H NMR (500 MHz, CDCl3):  = 8.26–8.21 (m, 2 H), 7.81–7.52 (m, 6 H),
7.45–7.27 (m, 4 H), 5.67 (s, 1 H), 4.74 (s, 2 H), 4.59 (dd, J = 10.6, 7.2 Hz,
1 H), 4.28 (dq, J = 14.2, 6.9 Hz, 3 H), 4.19–4.04 (m, 2 H), 1.25 (t, J = 7.1
Hz, 3 H).
13C NMR (126 MHz, CDCl3):  = 167.55 (CO), 156.33 (CO), 148.85
(CNO2), 143.90 (2 C)*, 143.09 (2 C), 141.59 (2 C)*, 141.44 (C), 141.00
(2 C), 128.07 (2 CH)*, 128.04 (2 CH), 127.77 (2 CH), 127.24 (2 CH),
125.42 (2 CH)*, 125.29 (2 CH), 123.63 (2 CH), 120.21 (2 CH)*, 120.17
(2 CH), 101.98 (CH), 68.23 (CH2), 66.17 (CH2), 62.51 (CH2), 57.34 (CH),
47.19 (CH), 14.26 (CH3); * signals of rotamers/diastereomers.
3-Ethyl 2-(9H-Fluoren-9-ylmethyl) (3S)-6-(4-Bromophenyl)-1,5,2-
dioxazinane-2,3-dicarboxylate (7)
IR (neat): 3674, 2986, 2900, 1719, 1449, 1304, 1209, 1084 cm–1
.
1H NMR (400 MHz, CDCl3):  = 7.79–7.20 (m, 12 H), 5.66 (s, 1 H), 4.80–
4.63 (m, 3 H), 4.49 (t, J = 9.9 Hz, 1 H), 4.34–4.23 (m, 3 H), 4.20–4.06
(m, 1 H), 1.26 (t, J = 7.1 Hz, 3 H).
HRMS (ESI): m/z [M + Na]+ calcd for C27H24N2NaO8: 527.1425; found:
527.1425.
13C NMR (101 MHz, CDCl3):  = 167.75 (CO), 156.42 (CO), 143.92 (C),
143.11 (C), 141.54 (C), 141.42 (C), 133.85 (C), 131.67 (2 CH), 128.42 (2
CH), 128.02 (2 CH)*, 127.97 (2 CH), 127.22 (2 CH), 125.66 (2 CH)*,
125.39 (2 CH), 124.21 (CBr), 120.16 (2 CH), 103.06 (CH), 68.35 (CH2),
66.12 (CH2), 62.41 (CH2), 57.26 (CH), 47.12 (CH), 14.27 (CH3); * signals
of rotamers/diastereomers.
3-Ethyl 2-(9H-Fluoren-9-ylmethyl) (3S)-6-Ethoxy-1,5,2-diox-
azinane-2,3-dicarboxylate (11)
IR (neat): 2674, 2986, 2901, 1741, 1450, 1355, 1176, 1057 cm–1
.
1H NMR (500 MHz, CDCl3):  = 7.86–7.61 (m, 4 H), 7.47–7.31 (m, 4 H),
5.43 (s, 1 H), 4.72–4.61 (m, 2 H), 4.57–4.31 (m, 2 H), 4.28 (q, J = 7.1 Hz,
2 H), 4.14–4.04 (m, 1 H), 3.88 (ddq, J = 44.5, 9.6, 7.1 Hz, 2 H), 1.37–
1.24 (m, 6 H); 1 H not observed.
HRMS (ESI): m/z [M + NH4]+ calcd for C27H28BrN2O6: 555.1125; found:
555.1118.
13C NMR (126 MHz, CDCl3):  = 167.46 (CO), 156.01 (CO), 143.85 (2
C)*, 143.31 (2 C), 141.50 (2 C)*, 141.44 (2 C), 128.02 (2 CH)*, 127.98 (2
CH), 127.26 (2 CH)*, 127.24 (2 CH), 125.50 (2 CH)*, 125.28 (2 CH),
120.15 (2 CH), 113.06 (CH), 68.54 (CH2), 64.90 (CH2), 63.20 (CH2),
62.45 (CH2), 47.07 (CH), 15.28 (CH3), 14.24 (CH3); * signals of rotam-
ers/diastereomers.; 1 C not observed.
3-Ethyl 2-(9H-Fluoren-9-ylmethyl) (3S)-6-[3,5-Bis(trifluorometh-
yl)phenyl]-1,5,2-dioxazinane-2,3-dicarboxylate (8)
IR (neat): 2970, 2901, 1727, 1450, 1277, 1175, 1132 cm–1
.
1H NMR (400 MHz, CDCl3):  = 8.22–7.21 (m, 9 H), 5.82 (br s, 1 H),
4.90–4.66 (m, 3 H), 4.58 (dd, J = 10.6, 7.4 Hz, 1 H), 4.38–4.24 (m, 3 H),
4.22–4.09 (m, 1 H), 1.33–1.20 (m, 3 H); 2 Harom not observed.
HRMS (ESI): m/z [M + NH4]+ calcd for C23H29N2O7: 445.1969; found:
445.1966.
13C NMR (101 MHz, CDCl3):  = 167.51 (CO), 141.54 (C), 132.02 (q, J =
33.9 Hz, 2 CF3), 128.08 (CH), 127.27 (CH), 125.30 (CH), 123.83 (CH),
120.15 (CH), 101.54 (CH), 68.71 (CH2), 66.24 (CH2), 62.62 (CH2), 57.55,
47.14 (CH), 14.24 (CH3); several C not assigned due to mixture of dias-
tereomers and rotamers.
19F NMR (377 MHz, CDCl3):  = –62.80.
HRMS (ESI): m/z [M + NH4]+ calcd for C29H27F6N2O6: 613.1768; found:
Fmoc Deprotection of Acid-Protected Monomer; General Proce-
dure
Piperidine (5.00 equiv) was added dropwise to Fmoc-protected
monomer (1.00 equiv) in CH2Cl2 (0.025 M). The reaction was stirred
for 1 h at r.t. The solution was diluted with CH2Cl2 and sat. aq NH4Cl
solution was added. The phases were separated and the aqueous layer
was extracted with CH2Cl2 (2 ×). The combined organic layers were
613.1769.
Georg Thieme Verlag Stuttgart · New York — Synthesis 2019, 51, A–K

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S. Baldauf, J. W. Bode
Paper
Synthesis
washed with brine and dried (Na2SO4). The drying agent was removed
by filtration and the solvent was evaporated. The residue was purified
by flash chromatography.
1H NMR (500 MHz, CDCl3):  = 7.45–7.27 (m, 5 H), 6.18 (s, 1 H), 5.29–
5.21 (m, 2 H), 4.21–4.03 (m, 2 H), 3.70 (d, J = 1.1 Hz, 3 H), 3.66 (t, J =
3.8 Hz, 1 H), 2.99–2.40 (m, 5 H).
13C NMR (126 MHz, CDCl3):  = 175.15 (CO), 169.67 (CO), 135.29 (C),
128.78 (2 CH), 128.66 (CH), 128.39 (2 CH)*, 128.37 (2 CH), 100.02
(C)*, 99.91 (C), 67.47 (CH2), 60.68 (CH2)*, 60.61 (CH2), 58.49 (CH)*,
58.41 (CH), 52.14 (CH)*, 52.12 (CH), 36.54 (CH2)*, 36.37 (CH2), 35.54
(CH2)*, 35.31 (CH2), 29.09 (CH)*, 28.96 (CH); * signals of rotamers/dia-
stereomers.
Ethyl (3S)-6-(4-Bromophenyl)-1,5,2-dioxazinane-3-carboxylate
(15)
IR (neat): 3674, 2986, 2901, 1732, 1490, 1369, 1210, 1043 cm–1
.
1H NMR (500 MHz, CDCl3):  = 7.50–7.45 (m, 2 H), 7.32–7.28 (m, 2 H),
6.86 (br s, 1 H), 5.80 (s, 1 H), 4.66 (dd, J = 11.4, 1.4 Hz, 1 H), 4.38–4.26
(m, 3 H), 3.52 (dd, J = 3.2, 1.4 Hz, 1 H), 1.34 (t, J = 7.1 Hz, 3 H).
HRMS (ESI): m/z [M + H]+ calcd for C16H20NO6: 322.1285; found:
322.1286.
13C NMR (126 MHz, CDCl3):  = 170.61 (CO), 135.50 (C), 131.53 (2 CH),
128.30 (2 CH), 123.58 (CBr), 101.88 (CH), 65.27 (CH2), 62.07 (CH2),
58.68 (CH), 14.39 (CH3).
HRMS (ESI): m/z [M + H]+ calcd for C12H15BrNO4: 316.0179; found:
316.0178.
Benzyl (S)-2-Bromo-5,9-dioxa-6-azaspiro[3.5]nonane-7-carboxyl-
ate (25)
IR (neat): 3674, 3296, 2987, 2900, 1738, 1454, 1282, 1198, 1065 cm–1
.
1H NMR (500 MHz, CDCl3):  = 7.44–7.33 (m, 5 H), 6.23 (s, 1 H), 5.33–
5.23 (m, 2 H), 4.33–4.13 (m, 2 H), 4.10 (ddd, J = 11.5, 3.4, 2.7 Hz, 1 H),
3.68 (dt, J = 7.7, 3.9 Hz, 1 H), 3.37–2.57 (m, 4 H).
13C NMR (126 MHz, CDCl3):  = 169.55 (CO)*, 169.45 (CO), 135.23 (C),
128.80 (2 CH), 128.71 (CH), 128.41 (2 CH)*, 128.39 (2 CH), 100.62 (C),
67.55 (CH2), 60.88 (CH2), 58.44 (CH)*, 58.41 (CH), 45.95 (CH2)*, 45.78
(CH2), 44.90 (CH2)*, 44.57 (CH2), 32.98 (CH)*, 32.75 (CH); * signals of
rotamers/diastereomers.
Ethyl (3S)-6-(4-Nitrophenyl)-1,5,2-dioxazinane-3-carboxylate (16)
IR (neat): 3674, 2987, 2901, 1734, 1522, 1348, 1214, 1044 cm–1
.
1H NMR (500 MHz, CDCl3):  = 8.22–8.18 (m, 2 H), 7.64–7.56 (m, 2 H),
6.94 (s, 1 H), 5.93 (s, 1 H), 4.70 (dt, J = 11.4, 1.4 Hz, 1 H), 4.37–4.29 (m,
3 H), 3.56 (dd, J = 3.3, 1.4 Hz, 1 H), 1.34 (t, J = 7.1 Hz, 3 H).
13C NMR (126 MHz, CDCl3):  = 170.45 (CO), 148.55 (CNO2), 142.82
(C), 127.64 (2 CH), 123.56 (2 CH), 100.98 (CH), 65.34 (CH2), 62.20
(CH2), 58.73 (CH), 14.40 (CH3).
HRMS (ESI): m/z [M + H]+ calcd for C14H17BrNO4: 342.0335; found:
342.0331.
HRMS (ESI): m/z [M + H]+ calcd for C12H15N2O6: 283.0925; found:
283.0927.
Benzyl Deprotection; General Procedure
Pd(OH)2/charcoal (20% on carbon wetted with ca. 50% water) (6%
m/m) was added to a solution of benzyl-protected acid (1.00 equiv) in
CH2Cl2/EtOH (1:4) at 0 °C. The mixture was stirred under H2 atmo-
sphere for 1 h while gradually warming up to r.t. The mixture was fil-
tered through a plug of celite and the solvent was evaporated.
Ethyl (3S)-6-Ethoxy-1,5,2-dioxazinane-3-carboxylate (17)
IR (neat): 3674, 2987, 2900, 1736, 1393, 1250, 1074, 1056 cm–1
1H NMR (500 MHz, CDCl3):  = 6.37 (d, J = 119.9 Hz, 1 H), 5.43 (dd, J =
11.2, 0.5 Hz, 1 H), 4.43–4.35 (m, 1 H), 4.26 (p, J = 7.2 Hz, 2 H), 4.09–
4.01 (m, 1 H), 3.89–3.66 (m, 3 H), 1.33–1.28 (m, 3 H), 1.28–1.23 (m, 3
H).
.
(S)-6-[(9H-Fluoren-9-ylmethoxy)carbonyl]-5,9-dioxa-6-
13C NMR (126 MHz, CDCl3):  = 169.44 (CO)*, 168.41 (CO), 111.02
(CH)*, 110.47 (CH), 62.92 (CH2)*, 62.69 (CH2), 62.63 (CH2)*, 62.46
(CH2), 61.97 (CH2)*, 61.89 (CH2), 57.65 (CH)*, 57.53 (CH), 15.16 (CH3),
14.28 (CH3)*, 14.26 (CH3); * signals of diastereomers.
HRMS (ESI): m/z [M + Na]+ calcd for C8H15NNaO5: 228.0842; found:
228.0842.
azaspiro[3.5]nonane-7-carboxylic Acid (26)
IR (neat): 3674, 2987, 2900, 1717, 1450, 1394, 1266, 1133, 1065 cm–1
1H NMR (500 MHz, CDCl3):  = 8.65 (br s, 1 H), 7.79–7.73 (m, 2 H),
7.64 (d, J = 45.5 Hz, 2 H), 7.44–7.37 (m, 2 H), 7.31 (tt, J = 7.4, 1.1 Hz, 2
H), 4.66 (dd, J = 10.5, 6.2 Hz, 1 H), 4.49 (s, 2 H), 4.38–4.22 (m, 2 H),
4.01 (s, 1 H), 2.44–2.18 (m, 4 H), 1.87 (dtd, J = 19.3, 12.1, 10.7, 7.0 Hz,
2 H).
.
13C NMR (126 MHz, CDCl3):  = 173.61 (CO), 156.39 (CO), 143.82 (2
C)*, 143.19 (2 C), 141.50 (2 C)*, 141.48 (2 C), 128.05 (2 CH)*, 127.98 (2
CH), 127.39 (2 CH)*, 127.17 (2 CH), 125.42 (2 CH)*, 125.08 (2 CH),
120.14 (2 CH), 105.24 (C), 68.62 (CH2), 60.08 (CH2), 56.89 (CH), 46.99
(CH), 32.60 (CH2), 30.25 (CH2), 11.47 (CH2); * signals of rotamers.
HRMS (ESI): m/z [M + Na]+ calcd for C22H21NNaO6: 418.1261; found:
418.1261.
Ethyl (S)-5,9-Dioxa-6-azaspiro[3.5]nonane-7-carboxylate (18)
IR (neat): 3674, 2987, 2901, 1733, 1451, 1375, 1280, 1079, 1020 cm–1
.
1H NMR (500 MHz, CDCl3):  = 4.26 (q, J = 7.2 Hz, 2 H), 4.07 (d, J = 4.4
Hz, 2 H), 3.67 (t, J = 4.4 Hz, 1 H), 2.44–2.14 (m, 4 H), 1.81–1.71 (m, 2
H), 1.30 (t, J = 7.1 Hz, 3 H); NH not observed.
13C NMR (126 MHz, CDCl3):  = 169.68 (CO), 103.05 (C), 61.79 (CH2),
60.59 (CH2), 58.37 (CH), 32.30 (CH2), 31.64 (CH2), 14.28 (CH3), 11.85
(CH2).
(S)-6-[(9H-Fluoren-9-ylmethoxy)carbonyl]-2-(methoxycarbonyl)-
5,9-dioxa-6-azaspiro[3.5]nonane-7-carboxylic Acid (27)
IR (neat): 3674, 2987, 2900, 1730, 1606, 1450, 1266, 1224, 1065 cm–1
HRMS (ESI): m/z [M + H]+ calcd for C9H16NO4: 202.1074; found:
202.1074.
.
1H NMR (400 MHz, CDCl3):  = 7.78–7.73 (m, 2 H), 7.72–7.49 (m, 2 H),
7.45–7.28 (m, 4 H), 4.70–4.25 (m, 5 H), 4.14–3.91 (m, 1 H), 3.71 (d, J =
20.1 Hz, 3 H), 3.10–2.25 (m, 4 H).
13C NMR (101 MHz, CDCl3):  = 174.71 (CO)*, 174.37 (CO), 172.75
(CO)*, 172.68 (CO), 158.09 (CO), 143.85 (2 C)*, 143.33 (2 C)*, 143.19 (2
C), 141.50 (2 C)*, 141.49 (2 C)*, 141.43 (2 C)*, 141.40 (2 C), 128.04 (2
7-Benzyl 2-Methyl (S)-5,9-Dioxa-6-azaspiro[3.5]nonane-2,7-dicar-
boxylate (24)
IR (neat): 3674, 2987, 2900, 2253, 1733, 1453, 1393, 1258, 1065, 903
cm–1
.
Georg Thieme Verlag Stuttgart · New York — Synthesis 2019, 51, A–K

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Synthesis
CH)*, 127.99 (2 CH), 127.95 (CH), 127.39 (2 CH)*, 127.37 (2 CH),
127.19 (2 CH)*, 127.06 (2 CH)*, 127.04 (2 CH), 125.61 (2 CH)*, 125.32
(2 CH)*, 125.25 (2 CH)*, 125.02 (2 CH), 120.16 (2 CH)*, 120.09 (2 CH),
101.88 (C)*, 101.65 (C), 68.97 (CH2)*, 68.34 (CH2), 60.52 (CH2)*, 60.42
(CH2), 52.30 (CH3), 47.06 (CH)*, 46.89 (CH), 36.54 (CH2)*, 36.37 (CH2),
34.60 (CH2)*, 34.20 (CH2), 28.79 (CH), 28.67 (C); * signals of rotam-
ers/diastereomers.
Ethyl N-[3-(4-Nitrophenyl)propanoyl]-L-serinate (14)
IR (neat): 3384, 2987, 2901, 1734, 1646, 1517, 1451, 1346, 1228, 1056
cm–1
1H NMR (500 MHz, MeOD):  = 8.19–8.13 (m, 2 H), 7.51–7.46 (m, 2 H),
4.48 (dd, J = 5.1, 4.2 Hz, 1 H), 4.18 (qd, J = 7.2, 2.2 Hz, 2 H), 3.88–3.71
(m, 2 H), 3.07 (td, J = 7.4, 1.3 Hz, 2 H), 2.65 (td, J = 7.5, 2.1 Hz, 2 H),
1.25 (t, J = 7.1 Hz, 3 H).
13C NMR (126 MHz, MeOD):  = 174.56 (CO), 171.76 (CO), 150.32 (C),
147.97 (CNO2), 130.61 (2 CH), 124.53 (2 CH), 62.80 (CH2), 62.46 (CH2),
56.27 (CH), 37.50 (CH2), 32.27 (CH2), 14.43 (CH3).
.
HRMS (MALDI/ESI): m/z [M + Na]+ calcd for C24H23NNaO8: 476.1316;
found: 476.1316.
(S)-2-Bromo-6-[(9H-fluoren-9-ylmethoxy)carbonyl]-5,9-dioxa-6-
azaspiro[3.5]nonane-7-carboxylic Acid (28)
HRMS (ESI): m/z [M + Na]+ calcd for C14H18N2NaO6: 333.1057; found:
333.1061.
IR (neat): 3674, 2987, 2900, 1717, 1450, 1408, 1283, 1264, 1139, 1067
cm–1
.
Benzyl N-(3-(4-Nitrophenyl)propanoyl)-L-serinate (23)
1H NMR (400 MHz, CDCl3):  = 9.23 (br s, 1 H), 7.82–7.73 (m, 2 H),
7.59 (dd, J = 24.6, 7.5 Hz, 2 H), 7.46–7.28 (m, 4 H), 4.67 (d, J = 5.9 Hz, 2
H), 4.36–4.21 (m, 2 H), 4.20–3.97 (m, 1 H), 3.96–3.85 (m, 1 H), 2.95 (d,
J = 15.7 Hz, 2 H), 2.70–2.45 (m, 2 H); 1 H not observed.
13C NMR (101 MHz, CDCl3):  = 173.11 (CO), 155.77 (CO), 143.71 (2
C)*, 143.08 (2 C), 141.55 (2 C)*, 141.51 (2 C), 128.09 (2 CH)*, 128.05 (2
CH), 127.37 (2 CH)*, 127.26 (2 CH), 125.05 (2 CH)*, 124.90 (2 CH),
120.21 (2 CH)*, 120.15 (2 CH), 102.49 (C), 68.01 (CH2), 60.71 (CH2),
47.16 (CH), 45.42 (CH2), 43.85 (CH2), 31.46 (CH); * signals of rotam-
ers/diastereomers; 1 C not observed.
IR (neat): 2939, 1737, 1643, 1515, 1454, 1343, 1190 cm–1
.
1H NMR (500 MHz, MeOD):  = 8.14–8.10 (m, 2 H), 7.47–7.43 (m, 2 H),
7.38–7.28 (m, 5 H), 5.22–5.13 (m, 2 H), 4.55 (dd, J = 5.2, 4.2 Hz, 1 H),
3.90–3.73 (m, 2 H), 3.09–2.99 (m, 2 H), 2.63 (td, J = 7.5, 2.3 Hz, 2 H).
13C NMR (126 MHz, MeOD):  = 174.64 (CO), 171.64 (CO), 150.28 (C),
147.95 (CNO2), 137.20 (C), 130.57 (2 CH), 129.54 (2 CH), 129.27 (CH),
129.11 (2 CH), 124.52 (2 CH), 68.00 (CH2), 62.75 (CH2), 56.35 (CH),
37.47 (CH2), 32.26 (CH2).
HRMS (ESI): m/z [M + Na]+ calcd for C19H20N2NaO6: 395.1214.1057;
+
Na]+ calcd for C22H20BrNNaO6:
found: 395.1218.
HRMS (MALDI/ESI): m/z [M
496.0366; found: 496.0367.
(S)-N-(2-amino-2-oxoethyl)-2-(2-((2S,5S)-6-hydroxy-5-(3-(4-ni-
trophenyl)propanamido)-3,4-dioxohexan-2-yl)hydrazineyl)-3-(4-
hydroxyphenyl)propanamide (32)
HRMS (ESI): m/z [M + Na]+ calcd for C26H32N6NaO9: 595.2123; found:
(S)-2-(tert-Butoxycarbonyl)-6-[(9H-fluoren-9-ylmethoxy)carbon-
yl]-5,9-dioxa-6-azaspiro[3.5]nonane-7-carboxylic Acid (29)
IR (neat): 3674, 2976, 2901, 1719, 1450, 1366, 1283, 1135, 1060 cm–1
1H NMR (400 MHz, CDCl3):  = 7.80–7.55 (m, 4 H), 7.44–7.28 (m, 4 H),
4.74–3.61 (m, 6 H), 3.03–2.01 (m, 5 H), 1.46 (d, J = 19.5 Hz, 9 H); OH
not observed.
.
595.2123.
HPLC (Shiseido Capcell Pak UG80 C18 column (4.6 × 250 mm), heated
to 60 °C, 10 to 90% CH3CN with 0.1% TFA in 20 min): tR = 19.8 min.
13C NMR (101 MHz, CDCl3):  = 173.54 (CO)*, 173.23 (CO), 173.10
(CO)*, 173.01 (CO), 156.42 (CO), 143.86 (2 C)*, 143.32 (2 C)*, 143.19 (2
C), 141.49 (2 C)*, 141.42 (2 C)*, 141.37 (2 C), 128.03 (2 CH)*, 127.96 (2
CH)*, 127.92 (2 CH), 127.41 (2 CH)*, 127.37 (2 CH)*, 127.19 (2 CH),
125.66 (2 CH)*, 125.34 (2 CH)*, 125.04 (2 CH), 120.16 (2 CH)*, 120.05
(2 CH), 101.97 (C), 101.65 (C), 81.06 (C)*, 81.01 (C), 68.91 (CH2)*,
68.34 (CH2), 60.44 (CH2)*, 60.38 (CH2), 57.20 (CH), 47.04 (CH), 46.88
(CH), 36.49 (CH2)*, 36.21 (CH2), 34.49 (CH2)*, 34.01 (CH2), 29.93 (CH)*,
29.70 (CH), 28.18 (3 CH3); * signals of rotamers/diastereomers.
Segment 36
HRMS (ESI): m/z [M]+ calcd for C263H418N70O81S2: 5917.0183; found:
5918.0125.
HPLC (Shiseido Capcell Pak UG80 C18 column (4.6 × 250 mm), heated
to 60 °C, 10 to 90% CH3CN with 0.1% TFA in 20 min): tR = 22.4 min.
Funding Information
HRMS (MALDI/ESI): m/z [M + Na]+ calcd for C27H29NNaO8: 518.1785;
This work was supported by the Swiss National Science Foundation
found: 518.1783.
(169451).
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KAHA Ligation; General Procedure
-Ketoacid 13 (1.20 equiv) was added to the free hydroxylamine (1.00
equiv) and dissolved in HFIP/AcOH (1:1, 30 mM) containing 1% water.
The mixture was heated to 45 °C for 12 h with monitoring by analyti-
cal HPLC. The crude mixture was directly purified by preparative
HPLC (Shiseido Capcell Pak C18 column, 50 × 250 mm) heated to 60 °C
using a gradient of 5 to 80% MeCN in Milipore-H2O containing 0.1%
(v/v) TFA over 35 min. HPLC monitoring of the KAHA ligation was car-
ried out using Shiseido Capcell Pak UG80 C18 column (4.6 × 250 mm),
heated to 60 °C, using a gradient of 10 to 90% MeCN in Milipore-H2O
containing 0.1% (v/v) TFA over 20 min.
Acknowledgment
We thank the MS and NMR services of the Laboratorium für Organ-
ische Chemie at ETH Zürich for analyses and Haewon Song for cor-
recting the manuscript.
Supporting Information
Supporting information for this article is available online at
https://doi.org/10.1055/s-0037-1611635.
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Georg Thieme Verlag Stuttgart · New York — Synthesis 2019, 51, A–K

K
S. Baldauf, J. W. Bode
Paper
Synthesis
References
(7) (a) Tokuyama, H.; Kuboyama, T.; Amano, A.; Yamashita, T.;
Fukuyama, T. Synthesis 2000, 1299. (b) Tokuyama, H.;
Kuboyama, T.; Fukuyama, T. Org. Synth. 2003, 80, 207.
(c) Medina, S. I.; Wu, J.; Bode, J. W. Org. Biomol. Chem. 2010, 8,
3405.
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