An Optimized Preparation of 1,1-Dimethylallyl Esters and Their Application to Solid-Phase Peptide Synthesis

Article abstract of DOI:10.1021/acs.joc.8b00658

A one-step preparation of 1,1-dimethylallyl (DMA) esters was optimized for the C-terminal protection of a range of Fmoc-protected amino acids. This preparation is not sensitive to the scale of reaction and affords the corresponding DMA esters in 70-99% yield with high regioselectivity. Additionally, these DMA-protected amino acids were used with the backbone amide linker (BAL) of Albericio and Barany and found to resist diketopiperazine formation during the synthesis of a series of tripeptide esters. C-terminal DMA protection is compatible with the BAL linkage and allows for standard Fmoc-based methods to be used throughout the synthesis.

Full text of DOI:10.1021/acs.joc.8b00658

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Article
An Optimized Preparation of 1,1-Dimethylallyl Esters
and Their Application to Solid-Phase Peptide Synthesis
Matthew Alexander Hostetler, and Mark Alfred Lipton
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00658 • Publication Date (Web): 25 Jun 2018
Downloaded from http://pubs.acs.org on June 25, 2018
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An Optimized Preparation of 1,1ꢀDimethylallyl Esters and Their Application to Solidꢀ
Phase Peptide Synthesis
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Matthew A. Hostetler and Mark A. Lipton*
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Department of Chemistry and Cancer Center, Purdue University
560 Oval Drive, West Lafayette, IN 479207-2084
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lipton@purdue.edu
TOC/Abstract Graphic:
Abstract
A oneꢀstep preparation of 1,1ꢀdimethylallyl (DMA) esters was optimized for the Cꢀterminal protection of
a range of Fmocꢀprotected amino acids. This preparation is not sensitive to the scale of reaction, and
affords the corresponding DMA esters in 70ꢀ99% yield with high regioselectivity. Additionally, these
DMAꢀprotected amino acids were used with the Backbone Amide Linker (BAL) of Albericio and Barany
and found to resist diketopiperazine formation during the synthesis of a series of tripeptide esters. Cꢀ
terminal DMA protection is compatible with the BAL linkage and allows for standard Fmocꢀbased
methods to be used throughout the synthesis.
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Introduction
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Cyclic and Cꢀterminal modified peptides are important classes of synthetic targets involved in drug
discovery,1–5 natural product synthesis,6–8 and chemical ligation studies.9–12 A major advance in preparing
such targets was achieved when the Backbone Amide Linker (BAL) was introduced by Albericio, Barany,
and coworkers in 1998.13 This strategy allows for free manipulation of both termini during solidꢀphase
peptide synthesis (SPPS) by anchoring the growing peptide to a backbone amide nitrogen atom. This
added degree of flexibility facilitates the preparation of cyclic peptides, peptide aldehydes, alcohols,
thioesters, and pꢀnitroanilides.13–16 In the preparation of cyclic peptides and in instances where the
modified Cꢀterminus is susceptible to nucleophilic hydrolysis (thioesters and pꢀnitroanilides), the Cꢀ
terminus must be orthogonally protected as an allyl ester throughout Fmocꢀbased SPPS. However, allyl
protection results in nearꢀquantitative formation of diketopiperazine (DKP) upon piperidineꢀpromoted
Fmoc cleavage at the dipeptide stage (Figure 1).13
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Figure 1. Undesired DKP formation in the BAL system (Ref. 13)
In general, steric bulk is known to reduce this unwanted side reaction, but such Cꢀterminal protecting
groups (e.g. tertꢀbutyl ester) are not compatible with the BAL strategy as the peptide is cleaved from the
linker under the highly acidic conditions needed to deprotect the ester.13,17–20 Methods have been
developed to reduce DKP formation when allyl esters are employed with BAL, but they require
unconventional synthetic steps before standard Fmocꢀbased SPPS protocols can be used (Figure 2).13–15 In
an effort to contribute a more direct approach to remedying this issue, work in our lab has focused on
developing the 1,1ꢀdimethylallyl (DMA) Cꢀterminal protecting group. This group combines the steric
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bulk of tertꢀbutyl esters with the mild deprotection conditions of allyl esters.21,22 DMA esters are
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compatible with BALꢀbased strategies and allow for standard Fmocꢀbased SPPS throughout the synthesis.
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Figure 2. Methods to reduce DKP formation in the BAL system. A) Ref. 13 & 14; B) Ref. 15
In two previous communications, we reported twoꢀstep21 and oneꢀstep22 preparations of DMAꢀ
protected amino acids. While both these original reports focused on DMA ester preparation, the ability of
DMA esters to resist DKP formation during BALꢀbased SPPS was not studied. In the present work, we
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wish to report the use of DMA esters as a means of reducing DKP formation in the BAL system while
using standard SPPS protocols throughout the synthesis (Figure 2). In the process of exploring these
studies, it was found that the original, oneꢀstep DMA ester preparation suffered from scaleꢀdependent
sensitivities. These observations motivated us to explore improvements to the original procedure and
these improvements are also discussed herein.
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Results and Discussion
The previously described oneꢀstep DMA ester preparation22 was not optimized, and adapted directly
from earlier work reported by Julia and coworkers.23 In that work, Julia and coworkers found that
catalytic quantities of copper(I) salts promoted reverse prenylation (ɣꢀsubstitution) of potassium
carboxylates with various allylic sulfonium salts, including prenyldimethylsulfonium tetrafluoroborate
(1).23 When these conditions were used, the DMA ester of FmocꢀThr(tBu)ꢀOH was obtained on a 0.25
mmol scale in 27% yield, whereas on a 1.0 mmol scale the ester was obtained in 74% yield (Table 1,
entry 1). Similar scaleꢀdependent inconsistencies in yield and regioselectivity were observed with other
amino acids as well (Table 1). This anomaly was especially severe in the case of FmocꢀGlu(tBu)ꢀOH
(Table 1, entry 5) where an almost complete reversal of regioselectivity (in favor of the undesired, αꢀ
substituted isomer) was observed on the 0.25 mmol scale. Based on these results, it became evident that
conditions adapted from Julia and coworkers may not be as general for the preparation of DMAꢀprotected
amino acids as we had previously thought.22 We therefore sought to develop a more robust set of
conditions that were less sensitive to variations in scale and were compatible with a range of amino acids
and side chain protecting groups.
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Table 1. Scale dependent variations in yield and regioselectivity using original DMA ester
preparationa
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0.25 mmol scale
1.0 mmol scale
entry
AA
yield, %b
ɣ / αc
yield, %
ɣ / α
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5
Thr(tBu)
Ala
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89
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89
100 / 0
65 / 35
94 / 6
74
94 / 6
98
89 / 11
88 / 12
100 / 0
100 / 0
Val
94d
90d
84d
Tyr(tBu)
Glu(tBu)
92 / 8
32 / 68
`a Conditions reported in Ref. 22 for DMA ester preparation; dry conditions. b Isolated yield of combined ester
products after filtration through silica pad. c ɣ / α product ratio was determined by 1H NMR integration. d Yield and
α / ɣ ratio obtained from Ref. 22
.
Improved preparation of sulfonium salt (1). To begin our optimization process, we first focused our
attention on the preparation of prenyldimethylsulfonium tetrafluoroborate (1). In our earlier work we
reported a procedure to prepare 1 as a pale brown solid.22 Although this material was found to be stable
for a period of months at ꢀ20 °C (by NMR), it was reported that freshly prepared material afforded the
best yields in the formation of DMAꢀprotected amino acids. We hypothesized that the colored impurity
may have been responsible for the salt’s decomposition over time. In an effort to improve the shelf life of
the sulfonium salt, we have developed a new and convenient preparation that affords 1 as a colorless,
crystalline solid in 94% yield (Scheme 1). As before, prenyl alcohol (2) is converted to 1 using a mixture
of dimethyl sulfide and fluoroboric acid. The previously used aqueous workup was replaced by the
removal of volatile impurities from the crude reaction mixture under reduced pressure followed by
precipitation of crystalline 1 using cold diethyl ether. Material prepared by this method has proven to be
stable at ꢀ20 °C for at least 8 months (by NMR) with no degradation of yield in the preparation of DMA
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esters observed over a minimum of 5 months (Supporting Information Table S1). It is noteworthy that this
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revised method can be used to produce 1 on a multigram scale without loss of yield or purity.
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Scheme 1. Preparation of prenyldimethylsulfonium tetrafluoroborate (1)
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Optimized preparation of DMAꢀprotected amino acids. With 1 in hand, we next explored
optimization of the preparation of DMAꢀprotected amino acids. As indicated in Table 2, FmocꢀThr(tBu)ꢀ
OH (3a) was chosen as the model substrate to probe optimal DMA esterification conditions on the 0.5
mmol scale. Initially, the copper source was explored (Table 2, entries 1–3). While CuI (Table 2, entry 3)
showed the most promise in terms of yield, an erosion in ɣꢀselectivity was observed. Despite its lower
yield (17%), CuBr promoted perfect ɣꢀselectivity (Table 2, entry 2). These results correlated well with the
initial observations of Julia and coꢀworkers23 and for this reason CuBr was chosen as the copper source
for the remainder of the optimization process.
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Table 2. Optimization of the DMA esterification of FmocꢀThr(tBu)ꢀOHa
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entry
CuX
CuCl
CuBr
CuI
CuBr
CuBr
CuBr
CuBr
CuBr
base
atmb
air
air
air
air
air
air
N2
yield of 4a, %c
ɣ / αd
87 / 13
100 / 0
91 / 9
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K2CO3
K2CO3
K2CO3
Li2CO3
Na2CO3
Cs2CO3
K2CO3
Na2CO3
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80
90
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94 / 6
100 / 0
28 / 72
100 / 0
100 / 0
N2
92 (95)e (93)f
a Reactions were carried out on the 0.5 mmol scale (unless otherwise noted) with 1.1 eq prenyl sulfonium salt (1),
1.1 eq base, 10 mol% CuX and 5 mL (0.1 M) CH2Cl2, 18ꢀ21 hrs. b Dry CH2Cl2 was used for reactions carried out
under N2 atm. c Isolated yield of product after filtration through silica pad. d ɣ / α product ratio was determined by 1H
NMR integration. e Yield of reaction carried out on the 0.25 mmol scale, 0.08 M CH2Cl2, ɣ / α: (99 / 1). f Yield of
reaction carried out on 1 mmol scale, 0.08 M CH2Cl2, ɣ / α: (96 / 4).
Though the mechanism for this transformation has not been established, it has been proposed to
proceed through the intermediacy of a reactive anionic copper(I) complex.23 We hypothesized that the
counterion to this complex might play a role in its reactivity. To probe this effect, we replaced potassium
carbonate with lithium, sodium, and cesium carbonate, respectively (Table 2, entries 2, 4–6). The use of
sodium carbonate afforded a substantial increase in the yield of 4a from 17% (Table 2, entry 2) to 80%
and proceeded without loss of ɣꢀselectivity (Table 2, entry 5). The use of cesium carbonate afforded 4a in
90% yield but with substantial loss of ɣꢀselectivity (Table 2, entry 6). The observed erosion in ɣꢀ
selectivity can likely be attributed to the cesium effect,24,25 whereby the cesium ion promotes a direct SN2
reaction between the cesium carboxylate and sulfonium salt. This SN2 process could compete with the
desired, copperꢀcatalyzed reverse prenylation and lead to the observed erosion in ɣꢀselectivity. The use of
an inert atmosphere (N2) and dry solvent with sodium carbonate as the base further improved the yield of
4a to 92% (Table 2, entry 8). Notably, no erosion of yield or ɣꢀselectivity was observed upon scaling
down (0.25 mmol) or up (1.0 mmol) under these conditions (Table 2, entry 8).
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With the optimized DMA esterification conditions in hand, we next examined the substrate scope by
subjecting a range of Fmocꢀprotected amino acids to the optimized conditions on the 0.25 mmol scale
(Table 3). The conditions furnished the corresponding DMA esters in 70–99% yield with high ɣꢀ
selectivity. The reactions were generally complete in 15 to 24 h, and in most cases the resulting esters
could be purified by silica pad filtration. As previously observed,22 amino acids containing nucleophilic
side chains require additional 1 to afford acceptable yields (3n). Additionally, the reaction was tested on
the 2.0 mmol scale with Ala and Val. In both instances, the reaction showed no sensitivity to scale and
resulted in high yields and regioselectivities (3c & 3d).
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Table 3. Synthesis of DMA esters from Fmocꢀprotected amino acids using optimized conditionsa
AA
Thr(tBu) (3a)
Gly (3b)
Ala (3c)
Val (3d)
yield of 4, %b
ɣ / αc
99 / 1
95
77
95 / 5
88 (93)d
84 (84)d
90
97 / 3 (98 / 2)d
98 / 2 (97 / 3)d
93 / 7
Leu (3e)
Tyr(tBu) (3f)
Ser(tBu) (3g)
Asn(Trt) (3h)
Cys(Trt) (3i)
Cys(PMB) (3j)
Pro (3k)
96
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77
98 / 2
96 / 4
96 / 4
96 / 4
96 / 4
92 / 8
Glu(tBu) (3l)
Lys(Boc) (3m)
Arg(Pbf) (3n)
80
90 / 10
79
94 / 6
56 (70)e
93 / 7 (96 / 4)e
a
Reactions were carried out on the 0.25 mmol scale (unless otherwise noted) with 1.1 eq
prenyl sulfonium salt (1), 1.1 eq Na2CO3, 10 mol% CuBr and 3.1 mL (0.08M) dry CH2Cl2,
b
15ꢀ24 h.
Isolated yield of product after filtration through silica pad or column
c
1
chromatography; see supporting information. ɣ / α product ratio was determined by H
NMR integration. d Reaction carried out on the 2.0 mmol scale. e Results obtained using 3.0
eq of 1.
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DMA as a Cꢀterminal protecting group for use in SPPS with the Backbone Amide Linker
(BAL).13 Having developed a robust preparation of DMAꢀprotected amino acids, we next sought to
explore their use in conjunction with SPPS using the BAL system. More specifically, we wished to study
the ability for DMA esters to resist undesired diketopiperazine (DKP) formation upon piperidineꢀ
promoted Fmoc deprotection at the dipeptide stage. To accomplish this, we employed a “dualꢀlinker”
system shown in Figure 3 where the peptideꢀBAL linkage would remain intact upon short exposure to the
dilute acid cleavage conditions required for the Sieber amide26,27 linkage. This strategy, which has been
described before,28 allows for rapid reaction analysis of resinꢀbound synthetic intermediates by UPLCꢀ
MS. All the products being detected possess the same UVꢀactive (254 nm) BAL chromophore which
facilitated uniform reaction component determination and crossꢀcomparison to integration at 214 nm.29
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Figure 3. “Dualꢀlinker” strategy showing compatible dilute acidic cleavage of Sieber amide linkage
The ortho backbone amide linker (oꢀBAL)30 was chosen for this study due to its ease of preparation
from commercially available starting material. The synthesis of oꢀBAL is shown in Scheme 2 and follows
a protocol similar to that originally described by Jensen and coꢀworkers.30 Orthoꢀselective demethylation
of 2,4,6ꢀtrimethoxybenzaldehyde (5) was accomplished using 1.0 M BBr3 in CH2Cl2 to afford oꢀphenol 6
in 86% yield. Cesium carbonateꢀpromoted alkylation of 6 with commercially available ethyl 5ꢀ
bromovalerate (7) furnished the corresponding ethyl ester 8 in 92% yield. Finally, saponification of 8 with
4 M aqueous NaOH–MeOH (1:1) provided oꢀPALdehyde (9) in 83% yield. Acid 9 was then loaded onto
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the solid support via HATUꢀmediated coupling with Sieber amide PS resin (10). After two rounds of
coupling, the remaining free amino groups were capped with Ac2Oꢀpyridine (3:2) to afford “dualꢀlinker”
11 (Scheme 3).
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Scheme 2. Synthesis of oꢀPALdehyde 930
Scheme 3. Synthesis of solidꢀsupported “dualꢀlinker” 11
Linker 11 was used to synthesize a series of tripeptides (Table 4) by the sequence of steps outlined in
Scheme 4. In each instance, the tripeptideꢀDKP ratio was determined by integration of UPLCꢀMS
chromatograms at 214 and 254 nm. Despite many attempts,31 the isolation and separation of Nꢀterminally
deprotected DMA amino esters from dibenzofulvene or its addition product proved difficult. To
circumvent these issues, we utilized a twoꢀstep reductive amination procedure starting with Fmocꢀ
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protected DMA esters. In this sequence, diethylamineꢀpromoted Fmoc deprotection was completed in the
presence of 1ꢀoctanethiol to scavenge the dibenzofulvene byproduct. The reaction was routinely complete
within 2 h and, following evaporation of excess diethylamine, afforded the corresponding amino DMA
esters. The resulting crude material was directly subjected to NaBH(OAc)3ꢀpromoted reductive amination
conditions with solid supported oꢀPaldehyde 11 to afford the corresponding BALꢀlinked benzylamines.
The resulting benzylamines were then acylated with preformed amino acid symmetric anhydrides to
afford resinꢀbound, Fmocꢀprotected dipeptides (12aꢀd). Fmoc deprotection with 20% piperidineꢀDMF (1
x 5 min, 1 x 10 min) followed by acylation with a preformed symmetric anhydride of FmocꢀGlu(tBu)ꢀOH
afforded Fmocꢀprotected tripeptides. Fmoc deprotection followed by Sieber amide cleavage with TFAꢀ
H2OꢀCH2Cl2 (2:1:97) provided BALꢀlinked tripeptides (13aꢀe) which were analyzed by UPLCꢀMS (Table
4). It is of note that we found DMA esters to be partially labile to Sieber amide cleavage conditions. For
this reason, both the DMA intact and the free acid were combined to compute the yields of tripeptide
products.
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Scheme 4. Synthesis of BALꢀlinked, DMAꢀprotected tripeptides (13aꢀd)
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Table 4. Tripeptide / DKP ratio during synthesis of BALꢀlinked DMAꢀprotected tripeptides
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Tripeptide
AA1
Gly
Ala
Leu
Ala
Ala
AA2
Ala
Ala
Ala
Leu
Ala
Cꢀterminal PG
DMA
Tripeptide / DKPa,b
83 / 17
13a
13b
13c
13d
13ed
DMA
DMA
70 / 30 (83 / 17)c
93 / 7
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DMA
82 / 18
94 / 6
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tBud
a Product ratios are relative, determined by UPLC integration at 214 nm and were crossꢀcompared with those at
254 nm. b DMA ester was partially labile to Sieber amide cleavage conditions and the combination of these two
products are represented in tripeptide values. c Tripeptide / DKP ratio obtained after 3 min piperidineꢀDMF
Fmoc deprotection at dipeptide stage. d Starting dipeptide was prepared using commercially available HꢀAlaꢀ
OtBu x HCl.
In their original report, Albericio and Barany found piperidineꢀpromoted Fmoc deprotection of Fmocꢀ
AlaꢀBALꢀGlyꢀOAllyl to result in quantitative DKP formation.13 We were pleased to find that by replacing
allyl ester with DMA ester (13a), an 83 / 17 tripeptide to DKP ratio was obtained. Intriguingly, the
slightly more hindered FmocꢀAlaꢀBALꢀAlaꢀODMA sequence (13b) was found to afford the most DKP of
all examples explored with a tripeptide to DKP ratio of 70 / 30. We were pleased to find that the ratio of
desired tripeptide 13b to DKP could be increased to 83 / 17 by reducing the duration of piperidine
treatment to 3 minutes at the dipeptide stage. While the purpose of this report was to test the limits of the
ability of Cꢀterminal DMA protection to resist DKP formation, we recommend shorter (2ꢀ3 min)
piperidine treatments during Fmoc deprotection at the dipeptide stage. To obtain a direct comparison
between tertꢀbutyl and DMA, the AlaꢀBALꢀAla tertꢀbutyl variant (13e) was also explored and found to
afford a 94 / 6 tripeptide to DKP ratio. Although tertꢀbutyl remains the most effective Cꢀterminal
protecting group for reducing DKP formation, it is not orthogonal to BAL and consequently not
applicable to the problem in question. As was shown in our previous report,21 DMA esters can be cleanly
removed by palladium(0) which has been wellꢀestablished13–15 as an orthogonal deprotection strategy for
use with the BAL. Furthermore, DMA esters are effective at resisting DKP formation, and offer an
alternative to the available methods for avoiding this undesired byproduct during BALꢀbased SPPS.13–15
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In summary, we have developed a robust preparation of DMAꢀprotected amino acids that is tolerant to
changes in scale and compatible with a wide range of Fmocꢀprotected amino acids. These Nꢀ and Cꢀ
terminally protected amino acids can be directly loaded onto the solid support by a twoꢀstep Fmoc
deprotection ꢀ reductive amination sequence with resinꢀbound PALdehyde. Cꢀterminal DMA protection
introduces a second semiꢀpermanent protecting group that is orthogonal to Fmocꢀbased SPPS, compatible
with the Backbone Amide Linker, and resistant to DKP formation.
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Experimental Section
General Information. Solvents were purified and dried on activated alumina columns prior to use.
Reactions where anhydrous conditions are noted were performed in glassware that was flame dried under
vacuum. Copper(I) bromide was purchased from Sigma Aldrich and purified by recrystallization from
1
hydrobromic acid and water. Carbonate salts were flame dried under vacuum prior to use. H NMR and
13C NMR spectra were recorded on Bruker DRXꢀ500, Bruker AVꢀIIIꢀ400 and Varian Mercuryꢀ300
spectrometers in CDCl3. Chemical shift (δ values) are reported in parts per million (ppm) and coupling
constants (J values) are reported in Hz. Infrared spectra were recorded on a Thermo Nicolet Nexus FTIR
using an ATR attachment. Thinꢀlayer chromatography (TLC) was performed using EMD Millipore 60 Å
silica gelꢀcoated, glassꢀbacked plates with F254 indicator. The silica gel used for flash chromatography
was SiliaFlash silica 230ꢀ400 mesh, 60 Å particle size. Melting points were measured using a Thomas
Hoover melting point apparatus and are uncorrected. UPLCꢀMS was performed on a Waters Acquity
UPLC® in conjunction with a Waters single quadrupole mass spectrometer using electrospray ionization
(ESI) and a photodiode array detector (measuring at 214 and 254 nm). A UPLC BEH C18 reverse phase,
1.7 µm, 2.1 x 50 mm column was used for separation. Low resolution mass spectrometry (MS) was
recorded on an Advion Expression CMS single quadrupole mass spectrometer, using a TLC plate reader
and electrospray ionization (ESI). High resolution mass spectra (HRMS) were recorded on a Sciex
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TripleTOF 5600 quadrupole timeꢀofꢀflight (TOF) mass spectrometer using nanoꢀelectrospray ionization
(nESI) and was performed independently by the McLuckey laboratory at Purdue University.
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Prenyldimethylsulfonium tetrafluoroborate (1).23 A solution of 3ꢀmethylꢀ2ꢀbutenꢀ1ꢀol (2.4 mL, 23
mmol) and dimethyl sulfide (5.2 mL, 71 mmol) in anhydrous CH2Cl2 (23 mL) was cooled in ice/salt
water bath (ꢀ10 °C) for 10 minutes. To this solution was added HBF4 (55 wt. % in diethyl ether, 3.1 mL,
23 mmol) dropwise under a N2 atmosphere. The reaction mixture was allowed to slowly warm to room
temperature and stirred for a total of 23 h, after which volatiles were removed under reduced pressure.
The resultant colorless oil was suspended in Et2O and cooled to 0°C for 15 min to afford a colorless
precipitate. Solids were vacuum filtered and washed with excess Et2O to afford 1 (4.76 g, 94%) as a
colorless, crystalline solid. mp 64ꢀ66 °C.
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General Preparation of 1,1ꢀdimethylallyl esters (4aꢀn). A mixture of carboxylic acid (0.250 mmol),
Na2CO3 (29.2 mg, 0.275 mmol) was taken up in dry CH2Cl2 (3.1 mL) and stirred for 5 min. To the
resulting mixture was added CuBr (3.6 mg, 0.025 mmol) followed by 1 (60.0 mg, 0.275 mmol) in
portions at rt. The mixture was stirred at rt, under an inert atmosphere. Following reaction completion
(15ꢀ24 h), solids were removed by filtration and the filtrate evaporated under reduced pressure. The crude
residue was purified by short SiO2 pad filtration (15ꢀ30% EtOAc/Hexanes). Solvent was removed under
reduced pressure, and the resulting residue was lyophilized twice to remove residual solvent and afford
pure 1,1ꢀdimethylallyl esters. Note: compounds which afforded byproducts (4i, 4j, 4l, 4n) were purified
by flash column chromatography in 20-60% EtOAc/Hexanes. *Corrected weight values factor trace
solvent impurities trapped in sample and were calculated via 1HꢀNMR integration.
FmocꢀThr(tBu) dimethylallyl ester (4a). Yield 95% (111 mg, corrected), colorless glass; Rf = 0.37
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(15% EA/Hex); ɣ / α: (99 / 1); H NMR (500 MHz, CDCl3) δ 7.77 (d, J = 7.5 Hz, 2H), 7.67 – 7.61 (m,
2H), 7.40 (tt, J = 7.5, 1.5 Hz, 2H), 7.32 (tt, J = 7.5, 1.5 Hz, 2H), 6.09 (dd, J = 17.5, 10.9 Hz, 1H), 5.54 (d,
J = 9.5 Hz, 1H), 5.25 – 5.17 (m, 1H), 5.09 (dd, J = 10.9, 0.8 Hz, 1H), 4.44 – 4.33 (m, 2H), 4.30 – 4.18 (m,
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2H), 4.16 (dd, J = 9.5, 2.3 Hz, 1H), 1.55 (s, 6H), 1.22 (d, J = 6.2 Hz, 3H), 1.19 (s, 9H); C NMR (125
MHz, CDCl3) δ 169.8, 156.7, 144.0, 143.9*, 142.0, 141.3, 127.7, 127.1, 125.2, 119.9, 113.2, 82.2, 73.9,
67.3, 67.1, 60.3, 47.2, 28.8, 26.4, 26.3*, 20.80 ppm; IR (neat) 3440, 2977, 2163, 1980, 1725, 1503, 1450,
1379, 1364, 1345, 1310, 1278, 1199, 1124, 1092, 1068, 986, 951, 920, 849; HRMS (nESIꢀTOF) m/Z:
[M+Na]+ Calcd for C28H35NO5Na 488.2413, Found 488.2414.
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FmocꢀGly dimethylallyl ester (4b).22 Yield 77% (71 mg), colorless solid; Rf = 0.41 (30%
EtOAc/Hexanes); mp 49ꢀ53 °C; ɣ / α: (95 / 5).
FmocꢀAla dimethylallyl ester (4c).22 Yield 88% (84 mg), colorless solid; Rf = 0.33 (20%
EtOAc/Hexanes); mp 76ꢀ77 °C; ɣ / α: (97 / 3).
FmocꢀVal dimethylallyl ester (4d).22 Yield 84% (86 mg, corrected), colorless glass; Rf = 0.37 (15%
EtOAac/Hexanes); ɣ / α: (98 / 2).
FmocꢀLeu dimethylallyl ester (4e). Yield 90% (95 mg, corrected), colorless glass; Rf = 0.34 (15%
EtOAc/Hexanes); ɣ / α: (93 / 7); 1H NMR (500 MHz, CDCl3) δ 7.77 (d, J = 7.5 Hz, 2H), 7.61 (d, J = 7.6
Hz, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.32 (td, J = 7.5, 1.1 Hz, 2H), 6.07 (dd, J = 17.5, 10.9 Hz, 1H), 5.21 (d,
J = 17.5 Hz, 1H), 5.17 (d, J = 8.7 Hz, 1H), 5.11 (d, J = 10.9 Hz, 1H), 4.39 (d, J = 7.2 Hz, 2H), 4.33 (td, J
= 9.0, 5.3 Hz, 1H), 4.23 (t, J = 7.1 Hz, 1H), 1.79 – 1.67 (m, 1H), 1.69 – 1.60 (m, 1H), 1.55 (s, 7H), 0.97
(dd, J = 6.6, 1.9 Hz, 6H); 13C NMR (125 MHz, CDCl3) δ 172.0, 155.9, 144.0, 143.8*, 141.9, 141.3,
127.7, 127.0, 125.1, 120.0, 113.2, 82.1, 66.9, 52.9, 47.2, 42.04, 26.4, 26.3*, 24.8, 22.9, 22.0*; IR (neat)
3334, 2957, 2158, 1715, 1525, 1468, 1450, 1413, 1381, 1366, 1334, 1260, 1221, 1162, 1119, 1046, 989,
927, 848; HRMS (nESIꢀTOF) m/z: [M+Na]+ Calcd for C26H31NO4Na 444.2151, Found 444.2165.
FmocꢀTyr(tBu) dimethylallyl ester (4f).22 Yield 96% (126 mg), colorless glassy solid; Rf = 0.35
(20% EA/Hex); ɣ / α: (98 / 2).
FmocꢀSer(tBu) dimethylallyl ester (4g). Yield 99% (112 mg, corrected), glass; Rf = 0.38 (20%
1
EtOAc/Hexanes); ɣ / α: (96 / 4); H NMR (500 MHz, CDCl3) δ 7.77 (d, J = 7.6 Hz, 2H), 7.63 (dd, J =
7.6, 4.2 Hz, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.32 (t, J = 7.5 Hz, 2H), 6.08 (dd, J = 17.5, 10.9 Hz, 1H), 5.65
(d, J = 8.8 Hz, 1H), 5.22 (d, J = 17.5 Hz, 1H), 5.10 (d, J = 10.9 Hz, 1H), 4.43 (dt, J = 9.0, 3.0 Hz, 1H),
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4.42 – 4.34 (m, 2H), 4.25 (t, J = 7.3 Hz, 1H), 3.84 (dd, J = 8.8, 2.9 Hz, 1H), 3.60 (dd, J = 8.7, 3.0 Hz,
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1H), 1.56 (d, J = 2.7 Hz, 6H), 1.18 (s, 9H); C NMR (125 MHz, CDCl3) δ 169.3, 156.1, 144.0, 143.9*,
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142.0, 141.3, 127.7, 127.1, 125.2, 119.9, 113.0, 82.2, 73.2, 67.1, 62.4, 54.9, 47.2, 27.4, 26.5, 26.4*; IR
(neat) 3444, 2975, 2159, 1724, 1505, 1477, 1450, 1392, 1381, 1364, 1339, 1296, 1234, 1197, 1163, 1127,
1102, 1083, 1059, 1022, 987, 922, 879, 850; HRMS (nESIꢀTOF) m/z: [M+Na]+ Calcd for C27H33NO5Na
474.2256, Found 474.2280.
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FmocꢀAsn(Trt) dimethylallyl ester (4h). Yield 89% (147 mg, corrected), white glassy solid; Rf =
0.38 (30% EtOAc/Hexanes); ɣ / α: (96 / 4); 1H NMR (500 MHz, CDCl3) δ 7.77 (d, J = 7.6 Hz, 2H), 7.60
(d, J = 7.5 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.35 – 7.22 (m, 13H), 7.21 – 7.15 (m, 6H), 6.67 (s, 1H), 6.12
(d, J = 8.7 Hz, 1H), 5.95 (dd, J = 17.5, 10.9 Hz, 1H), 5.14 (d, J = 17.4 Hz, 1H), 5.02 (dd, J = 10.9, 0.8 Hz,
1H), 4.53 (dt, J = 8.9, 4.3 Hz, 1H), 4.41 (dd, J = 10.4, 7.1 Hz, 1H), 4.28 (dd, J = 10.4, 7.5 Hz, 1H), 4.21
(t, J = 7.2 Hz, 1H), 3.09 (dd, J = 16.1, 4.4 Hz, 1H), 2.84 (dd, J = 16.1, 4.2 Hz, 1H), 1.45 (d, J = 9.1 Hz,
6H); 13C NMR (125 MHz, CDCl3) δ 169.6, 169.2, 156.3, 144.3, 144.0, 143.8*, 142.1, 141.3, 128.7,
128.0, 127.7, 127.2, 127.1, 125.3, 125.2*, 119.9, 112.9, 82.6, 70.9, 67.2, 51.2, 47.1, 38.6, 26.3, 26.1*; IR
(neat) 3328, 3058, 1728, 1491, 1448, 1413, 1380, 1365, 1331, 1215, 1157, 1125, 1081, 1035, 1002, 934,
903, 848; HRMS (nESIꢀTOF) m/z: [M+Na]+ Calcd for C43H40N2O5Na 687.2835, Found 687.2879.
FmocꢀCys(Trt) dimethylallyl ester (4i). Yield 87% (143 mg), colorless, glassy solid; Rf = 0.32 (15%
EtOAc/Hexanes); ɣ / α: (96 / 4); 1H NMR (500 MHz, CDCl3) δ 7.78 (dd, J = 7.7, 3.5 Hz, 2H), 7.62 (d, J
= 7.5 Hz, 2H), 7.44 – 7.38 (m, 8H), 7.34 – 7.27 (m, 8H), 7.24 – 7.18 (m, 3H), 6.04 (dd, J = 17.5, 10.9 Hz,
1H), 5.31 (d, J = 8.5 Hz, 1H), 5.17 (d, J = 17.5 Hz, 1H), 5.09 (d, J = 10.9 Hz, 1H), 4.36 (d, J = 7.4 Hz,
2H), 4.32 (dt, J = 9.5, 5.2 Hz, 1H), 4.24 (t, J = 7.3 Hz, 1H), 2.65 (dd, J = 12.2, 6.0 Hz, 1H), 2.58 (dd, J =
12.2, 4.6 Hz, 1H), 1.52 (s, 6H); 13C NMR (125 MHz, CDCl3) δ 169.2, 155.5, 144.3, 143.9, 143.8*, 141.7,
141.3, 129.5, 128.0, 127.7, 127.1, 126.8, 125.2, 120.0, 113.3, 82.9, 67.1, 66.8, 53.3, 47.1, 34.4, 26.3; IR
(neat,) 3409, 3059, 2162, 1980, 1723, 1596, 1492, 1447, 1413, 1380, 1365, 1342, 1207, 1157, 1123,
1081, 1049, 1001, 927, 846; HRMS (nESIꢀTOF) m/z: [M+Na]+ Calcd for C42H39NO4SNa 676.2498,
Found 676.2522.
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FmocꢀCys(PMB) dimethylallyl ester (4j). Yield 90% (120 mg, corrected), colorless glass; Rf = 0.36
(20% EtOAc/Hexanes); ɣ / α: (96 / 4); 1H NMR (500 MHz, CDCl3) δ 7.77 (dd, J = 7.6, 2.6 Hz, 2H), 7.62
(dd, J = 7.6, 4.0 Hz, 2H), 7.40 (td, J = 7.5, 3.5 Hz, 2H), 7.31 (tdd, J = 7.5, 2.7, 1.1 Hz, 2H), 7.22 (d, J =
8.6 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 6.07 (dd, J = 17.5, 10.9 Hz, 1H), 5.54 (d, J = 7.9 Hz, 1H), 5.20 (d, J
= 17.5 Hz, 1H), 5.11 (d, J = 10.9 Hz, 1H), 4.52 (dt, J = 7.9, 5.2 Hz, 1H), 4.45 – 4.35 (m, 2H), 4.24 (t, J =
7.2 Hz, 1H), 3.77 (s, 3H), 3.70 (s, 2H), 2.92 (dd, J = 13.8, 4.7 Hz, 1H), 2.83 (dd, J = 13.8, 5.8 Hz, 1H),
1.55 (s, 6H); 13C NMR (125 MHz, CDCl3) δ 169.5, 158.8, 155.7, 143.9, 143.8*, 141.6, 141.3, 130.1,
129.6, 127.7, 127.1, 125.1, 120.0, 114.0, 113.5, 83.0, 67.2, 55.3, 54.0, 47.1, 36.2, 33.7, 26.3; IR (neat)
3335, 2934, 2159, 1720, 1610, 1584, 1511, 1465, 1450, 1414, 1380, 1342, 1302, 1244, 1209, 1176, 1158,
1123, 1034, 996, 926, 832; HRMS (nESIꢀTOF) m/z: [M+Na]+ Calcd for C31H33NO5SNa 554.1977, Found
554.1992.
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FmocꢀPro dimethylallyl ester (4k).22 Yield 77% (78 mg, corrected), pale yellow glass; Rf = 0.31
(25% EtOAc/Hexanes); ɣ / α: (92 / 8).
FmocꢀGlu(tBu) dimethylallyl ester (4l).22 Yield 80% (100 mg), colorless glass; Rf = 0.34 (20%
EtOAc/Hexanes); ɣ / α: (90 / 10).
FmocꢀLys(Boc) dimethylallyl ester (4m). Yield 79% (106 mg), colorless, amorphous solid; Rf =
0.37 (30% EtOAc/Hexanes); mp 42ꢀ43 °C; ɣ / α: (94 / 6); 1H NMR (500 MHz, CDCl30) δ 7.77 (d, J = 7.5
Hz, 2H), 7.60 (d, J = 7.5 Hz, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.32 (tt, J = 7.4, 1.3 Hz, 2H), 6.07 (dd, J =
17.5, 10.9 Hz, 1H), 5.37 (d, J = 8.4 Hz, 1H), 5.21 (d, J = 17.5 Hz, 1H), 5.11 (d, J = 10.9 Hz, 1H), 4.56 (s,
1H), 4.44 – 4.34 (m, 2H), 4.29 (td, J = 7.9, 5.0 Hz, 1H), 4.22 (t, J = 7.0 Hz, 1H), 3.12 (d, J = 7.9 Hz, 2H),
1.91 – 1.80 (m, 1H), 1.73 – 1.65 (m, 1H), 1.64 (d, J = 2.0 Hz, 1H), 1.54 (s, 6H), 1.54 – 1.48 (m, 2H), 1.43
(s, 9H), 1.43 – 1.30 (m, 1H); 13C NMR (125 MHz, CDCl3) δ 171.2, 156.0, 155.9, 143.9, 143.8*, 141.8,
141.3, 127.7, 127.1, 125.1, 120.0, 113.4, 82.4, 79.2, 67.0, 54.0, 47.2, 40.2, 32.4, 29.6, 28.4, 26.4, 26.3,
22.3*; IR (neat) 3339, 2977, 2934, 2161, 1694, 1514, 1479, 1450, 1381, 1365, 1248, 1165, 1126, 1082,
1043, 928, 853; HRMS (nESIꢀTOF) m/z: [M+Na]+ Calcd for C31H40N2O6Na 559.2784, Found 559.2801.
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FmocꢀArg(Pbf) dimethylallyl ester (4n).22 The general procedure for the synthesis of 1,1ꢀ
dimethylallyl esters was followed with the use of FmocꢀArg(Pbf)ꢀOH (162 mg, 0.250 mmol), Na2CO3
(29.2 mg, 0.275 mmol), CuBr (3.6 mg, 0.025 mmol), and sulfonium salt 1 (180 mg, 0.83 mmol). Flash
column chromatography with 60% EtOAc/Hexanes afforded 4n as a colorless glassy solid (126 mg,
70%). Rf = 0.43 (60% EtOAc/Hexanes); ɣ / α: (96 / 4).
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2ꢀhydroxyꢀ4,6ꢀdimethoxybenzaldehyde30 (6). A solution of 2,4,6ꢀTrimethoxybenzaldehyde (5) (2.00
g, 10.2 mmol) in dry CH2Cl2 (51 mL) was cooled in an ice bath under an inert atmosphere for 10 min. To
this mixture was added BBr3 (1.0 M in CH2Cl2, 10.2 mL, 10.2 mmol) dropwise. The resulting deep red
solution was stirred at 0°C for 1 h, then warmed to rt and stirred until reaction completion was shown by
TLC (2 h). H2O (51 mL) was slowly added and the resulting mixture stirred for an additional 30 min. The
organic layer was removed and the aqueous layer was extracted with CH2Cl2 (3 x 50 mL). The combined
organic extracts were washed with brine (100 mL), dried over Na2SO4, filtered and evaporated under
reduced pressure. Colored impurities were removed by short SiO2 pad filtration (30% EtOAc/Hexanes) to
afford 6 as an offꢀwhite solid (1.61 g, 86%). Rf = 0.38 (25% EtOAc/Hexanes); mp 68ꢀ70 °C (lit.32 63ꢀ66
°C).
Ethyl 5ꢀ(2ꢀFormylꢀ3,5ꢀdimethoxyphenoxy)pentanoate (8).30 Cs2CO3 (4.20 g, 12.8 mmol) was added
to a solution of 6 (1.17 g, 6.42 mmol) in dry DMF (4.3 mL) at rt. Ethyl 5ꢀbromovalerate (2.00 mL, 12.8
mmol) was added dropwise, and this mixture was heated to 60 °C until reaction completion was shown by
TLC (4 h). The reaction was allowed to cool to rt, then diluted with H2O (8 mL). The mixture was
extracted with CH2Cl2 (4 x 15 mL) and the combined organic extracts were washed with brine (5 x 50
mL), dried over Na2SO4, filtered and evaporated under reduced pressure. Purification by flash
chromatography (60% EA/Hex) afforded 8 as a pale yellow solid (1.84 g, 92%). Rf = 0.35 (60%
EA/Hex); mp 44 – 45 °C (lit.30 44 – 45 °C).
5ꢀ(2ꢀFormylꢀ3,5ꢀdimethoxyphenoxy)pentanoic Acid (oꢀPALdehyde) (9).30,33 To a solution of 8
(1.74 g, 5.61 mmol) in MeOH (11.2 mL) was added 4 M NaOH (11.2 mL, 44.9 mmol) dropwise and the
resulting mixture stirred for 1.5 h at room temperature. MeOH was removed under reduced pressure and
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resulting aqueous phase extracted with EtOAc (2 x 15 mL) then acidified to pH ~1 with concentrated
HCl. The acidified aqueous phase was extracted with EtOAc (2 x 50 mL) and the combined organic
extracts were washed with brine (2 x 100 mL), dried over Na2SO4, filtered and evaporated under reduced
pressure to afford 9 as a yellow solid (1.31 g, 83%). mp 102–102.5 °C (lit.33 103–104 °C).
Cleavage condition A (Sieber Amide cleavage): An aliquot (~1 mg) of resinꢀbound peptide was
treated with 200 µL TFA/H2O/CH2Cl2 (2:1:97) and the mixture agitated for 5 min at rt. Following this
time period, volatiles were removed by N2 stream. The resulting residue was dissolved in 200 µL MeOHꢀ
H2O (1:1), filtered and analyzed by UPLCꢀMS. Note: Product / DKP ratio was determined by integrating
214 and 254 nm peaks and are reported as relative percentages.
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Standard wash protocol: DMF (3 x 1 mL), CH2Cl2 (3 x 1 mL), MeOH (3 x 1 mL) then CH2Cl2 (3 x 1
mL).
oꢀPALdehydeꢀSieberꢀPS “Dualꢀlinker” (11). FmocꢀSieber Amide Resin (10) (200 mg, 0.7 mmol/g,
1% DVB) was swelled in DMF for 30 min, drained, and washed with piperidineꢀDMF (1:4, 1 x 1 mL, 1 x
1.5 mL 30 min). The mixture was drained then washed with standard wash protocol affording a positive
Kaiser ninhydrin test.34 The resin was then washed with DMF (3 x 1 mL) and a mixture of oꢀPALdehyde
(9) (158 mg, 0.560 mmol) and HATU (213 mg, 0.560 mmol) in DMF (1.4 mL) was added DIEA (200 µL,
1.12 mmol). This mixture added to the resin after a 5 min preactivation period. After 2 h, resin was
washed by the standard wash protocol and coupling repeated. After the coupling was repeated for a
second time, the resin afforded a slightly positive Kaiser ninhydrin test.34 Remaining free amino groups
were capped by agitating resin with 1.5 mL Ac2Oꢀpyridine (3:2) mixture for 30 min, after which, the
Kaiser ninhydrin test34 was negative. The resin was washed using standard wash protocol, then MeOH
and dried under reduced pressure.
General procedure for SPPS of DMAꢀprotected tripeptides (13aꢀd). To a solution of FmocꢀAA1ꢀ
ODMA (0.14 mmol) in CH3CN (0.28 mL) was added 1ꢀoctanethiol (120 µL, 0.70 mmol) then
diethylamine (140 µL, 1.4 mmol) dropwise. The reaction was stirred at rt for 2 h, at which point volatiles
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were removed under reduced pressure. The crude residue was coꢀevaporated with CH3CN (3 x 5 mL) to
afford crude free amines of DMA esters that were used without further purification.
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NaBH(OAc)3 (29.7 mg, 0.140 mmol) was added to the crude amino DMA esters and the mixture taken
up in AcOHꢀDMF (1% solution, 0.3 mL). The resultant mixture was agitated to promote solubility and
added directly to oꢀPALdehydeꢀSieberꢀPS resin (11) (20.0 mg, 0.7 mmol/g, 0.014 mmol) that was
prewashed with DMF. The reaction mixture was agitated for 18.5 h, after which the solvent was removed
and the resin washed with water, followed by the standard wash protocol, then DMF (3 x 1 mL). The
reaction was agitated with piperidineꢀDMF (1:4, 1.5 mL, 5 min) then washed once more with the
standard wash protocol to give a positive chloranil test,35 affording Hꢀ(oꢀBALꢀSieberꢀPS)ꢀAA1ꢀODMA.
A separate flameꢀdried flask was charged with FmocꢀAA2ꢀOH (0.14 mmol) and dry CH2Cl2 (0.14 mL)
and symmetric anhydride formation was initiated by addition of DIPCDI (11 µL, 0.07 mmol) dropwise at
rt. The mixture was allowed to stand for 15 min and the resulting solid was solubilized by addition of 0.14
mL DMF. This solution was added directly to the resin and the reaction agitated for 2 h followed by the
standard wash protocol. This procedure was repeated twice to afford FmocꢀAA2ꢀ(oꢀBALꢀSieberꢀPS)ꢀ
AA1ꢀODMA, 12aꢀd (negative chloranil test).35
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The Nꢀterminal Fmoc protecting group was removed from the resinꢀbound peptide by agitation with
piperidineꢀDMF (1:4; 1 x 5 min, 1 x 10 min) followed by the standard wash protocol to afford a positive
Kaiser ninhydrin test.34 Acylation of the resulting amine intermediate was achieved by treatment with a
preꢀformed symmetric anhydride of FmocꢀGlu(tBu)ꢀOH (5 equiv), prepared using the procedure
described above, to afford FmocꢀGlu(tBu)ꢀAA2ꢀ(oꢀBALꢀSieberꢀPS)ꢀAA1ꢀODMA. The Nꢀterminal Fmoc
group was removed by agitation with piperidineꢀDMF (1:4; 1 x 5 min, 1 x 20 min) to afford HꢀGlu(tBu)ꢀ
AA2ꢀ(oꢀBALꢀSieberꢀPS)ꢀAA1ꢀODMA and a positive Kaiser ninhydrin test.34 Resin cleavage was
achieved using cleavage condition A to afford 13aꢀd and product ratio was determined by UPLCꢀMS.
HꢀGlu(tBu)ꢀAlaꢀ(oꢀBALꢀNH2)ꢀGlyꢀODMA (13a). UPLCꢀMS (5→95, CH3CNꢀH2O, 10 min
gradient): Rt 3.15 min, 17% DKP, MS (ESI) calcd for C19H27N3O6: 393.2, Found 394.4 [M+H]+; Rt 3.58
min, 61% DMA deprotected product, MS (ESI) calcd for C28H44N4O10: 596.3, Found 597.6 [M+H]+; Rt
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4.82 min, 22% DMA esterified product, MS (ESI) calcd for C33H52N4O10: 664.4, Found 665.6 [M+H]+.
83% Desired product, 17% DKP.
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HꢀGlu(tBu)ꢀAlaꢀ(oꢀBALꢀNH2)ꢀAlaꢀODMA (13b). UPLCꢀMS (25→40, CH3CNꢀH2O, 5 min
gradient): Rt 1.02 min, 30% DKP, MS (ESI) calcd for C20H29N3O6: 407.2, Found 408.3 [M+H]+; Rt 1.42
and 1.67 min, 50% (total) DMA deprotected product, MS (ESI) calcd for C29H46N4O10: 610.3, Found
611.4 [M+H]+; Rt 4.42 and 4.60 min, 20% (total) DMA esterified product, MS (ESI) calcd for
C34H54N4O10: 678.4, Found 679.4 [M+H]+. 70% desired product, 30% DKP. Note: an 83 / 17 tripeptide to
DKP ratio was obtained from short (3 min) 20% piperidineꢀDMF Fmoc deprotection at dipeptide stage.
This deprotection was followed by a brief washing cycle (3 x DMF, 3 x CH2Cl2, 1 x MeOH, 1 x CH2Cl2,
1 x DMF), and the remaining procedure for tripeptide formation and analysis was repeated as described in
the general procedure above (Supporting Information Figure S35).
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HꢀGlu(tBu)ꢀAlaꢀ(oꢀBALꢀNH2)ꢀLeuꢀODMA (13c). UPLCꢀMS (30 iso 1 min→50, CH3CNꢀH2O, 5
min gradient): Rt 1.85 min, 7% DKP, MS (ESI) calcd for C23H35N3O6: 449.3, Found 450.5 [M+H]+; Rt
2.00 and 2.25 min, 56% (total) DMA deprotected product, MS (ESI) calcd for C32H52N4O10: 652.4, Found
653.6 [M+H]+; Rt 4.53, 37% DMA esterified product, MS (ESI) calcd for C37H60N4O10: 720.4, Found
721.6 [M+H]+. 93% desired product, 7% DKP.
HꢀGlu(tBu)ꢀLeuꢀ(oꢀBALꢀNH2)ꢀAlaꢀODMA (13d). UPLCꢀMS (25 iso 1 min→50, CH3CNꢀH2O, 5
min gradient): Rt 2.78 min, 18% DKP, MS (ESI) calcd for C23H35N3O6: 449.3, Found 450.4 [M+H]+; Rt
2.95 and 3.25 min, 48.3% (total) DMA deprotected product, MS (ESI) calcd for C32H52N4O10: 652.4,
Found 653.5 [M+H]+; Rt 4.50, 4.65, 4.77, 4.87 min, 33.4% DMA esterified product, MS (ESI) calcd for
C37H60N4O10: 720.4, Found 721.5 [M+H]+. 82% desired product, 18% DKP.
HꢀGlu(tBu)ꢀAlaꢀ(oꢀBALꢀNH2)ꢀAlaꢀOtBu (13e). A mixture of HꢀAlaꢀOtBu x HCl (25.4 mg, 0.14
mmol, 10 eq) and NaBH(OAc)3 (29.7 mg, 0.14 mmol, 10 eq) was taken up in 0.3 mL of 1% AcOHꢀDMF
and this mixture was added directly to oꢀPALdehydeꢀSieberꢀPS resin (11) (20 mg, 0.7 mmol/g, 0.014
mmol). The reaction mixture was agitated for 18 h, after which the resin was filtered and washed with
water, followed by the standard wash protocol, then DMF (3 x 1 mL). The reaction was agitated with
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piperidineꢀDMF (1:4, 1.5 mL, 5 min) then washed once more with the standard wash protocol to give a
positive chloranil test,35 and afford Hꢀ(oꢀBALꢀSieberꢀPS)ꢀAlaꢀOtBu. The remaining synthetic steps were
repeated as described in the general procedure above. UPLCꢀMS (15→95, CH3CNꢀH2O, 5 min gradient):
Rt 1.82 min, 6% DKP, MS (ESI) calcd for C20H29N3O6: 407.2, Found 408.4 [M+H]+. Rt 2.50 min, 94%
desired product, MS (ESI) calcd for C33H54N4O10: 666.4, Found 667.7 [M+H]+. 94% desired product, 6%
DKP.
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Supporting Information
1H NMR stability studies for compound 1, 1H and 13C NMR for DMA esters 4aꢀn, 1H NMR for
compounds 6, 8 & 9, and UPLCꢀMS chromatograms for compounds 13aꢀe, Table S1: The effect of
sulfonium salt (1) age on the yield of DMA esterification.
Acknowledgments
We would like to thank David Foreman of the McLuckey Laboratory at Purdue for providing HRMS data
for this project.
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