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Synlett 2025; 36(01): 55-58
DOI: 10.1055/a-2306-9316
letter

Improved Synthesis of Pseudoproline and DMB Dipeptide Carboxylic Acids

Wyatt C. Powell
,
Kelly Johnson
,
Peyton Tran
,
Ruiheng Jing
,
Maciej A. Walczak∗

This work was supported by the National Institutes of Health (R01GM138682).
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Abstract

We report a mild method for the preparation of Fmoc-protected dipeptides containing a 2,4-dimethoxybenzyl (DMB) or 2-hydroxy-4-methoxybenzyl (HMB) group or modified as pseudoprolines. To minimize the loss of the Fmoc protection, we optimized the saponification conditions and included a calcium additive that protected the other base-sensitive functionalities and improved the yield of the free acid (36–82%). The reaction requires a combination of CaCl2 and NaOH in a mixture of iPrOH and water at room temperature.


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Key words

amino acids - dipeptides - saponification - deprotection - calcium
Zoom Image
Scheme 1 Overview of pseudoproline and DMB motifs in peptide synthesis and natural products

Modern preparative peptide and protein chemistries rely on ready access to protected amino acids and their derivatives.[1] Among various commercially available synthons, dipeptides constitute an important class of building blocks that are useful for the preparation of oligopeptide sequences with inherent propensities to aggregate and self-assemble. In this context, pseudoprolines (ΨPro) represent a valuable class of reagents derived from serine, threonine, and cysteine formed via condensation with an aldehyde or a ketone (Scheme [1]A).[2] [3] Their synthesis and applications have become a major focus of research due to their ability to influence peptide properties, particularly their solubility,[3,4] and can assist with macrocyclization.[5] Some notable recent applications include the synthesis of GLP-1 receptor agonists,[6] liraglutide,[7] islet amyloid polypeptide,[8] granulocyte colony-stimulating factor,[9] ubiquitin,[10] and tau protein.[11] In the same category, 2,4-dimethoxybenzyl (DMB) and 2-hydroxy-4-methoxybenzyl (HMB) protected dipeptides are also used to prevent undesired aspartimide cyclizations (Scheme [1]B). A special feature of pseudoprolines is their ability to disrupt secondary structure formation during solid-support peptide synthesis, a key feature for enhancing solubility (Scheme [1]C). Substituted pseudoprolines, such as trifluoromethyl pseudoprolines (CF3), also draw interest for their configurational and hydrolytic stability.[12] These substituted pseudoprolines serve as effective proline surrogates, notably in studying proline cis-trans isomerization.[13] ΨPro moieties are also found embedded in various naturally occurring and biologically active molecules. As illustrated in Scheme [1]D, the ΨPro component plays a critical role as a bicyclic structure within the indole alkaloid cadambinic acid,[14] serves as an inhibitor in endothelin converting enzyme TMC-66,[15] and is a key part in the β-lactamase inhibitor clavulanic acid.[16] Additionally, the Prothz group, a variant of ΨPro, is essential for the architecture of penicillins, particularly for creating the bicyclic framework that integrates with the β-lactam ring. The broad spectrum of these biological uses highlights the importance of pseudoprolines and emphasizes the demand for efficient syntheses.

Various pseudoproline and DMB dipeptides are known, but they are relatively expensive. Several strategies have been developed to access these building blocks, which include a direct fragment condensation with free carboxylic acids at the C-terminus[17] or dipeptide coupling with the C-terminal amino acid protected as benzyl, allyl, or methyl esters.[18]

The most common synthon of pseudoprolines and DMB-dipeptide is a carboxylic acid with an N-terminal Fmoc group that can be easily incorporated into the solid-support peptide synthesis. Although these amino acid building blocks are commercially available, their relatively high cost poses a significant challenge for large-scale solid-support reactions, which necessitate an excess of reagents. Here, we report a straightforward and scalable preparation of Fmoc-protected dipeptides including pseudoproline and DMB peptides using mild saponification conditions that retain the Fmoc group and significantly increase the overall yield.

Simple saponification of Fmoc-protected amino acids and peptides with aqueous LiOH or NaOH inevitably leads to the loss of the Fmoc group. Although the extent of ester hydrolysis can be reduced by adjusting the concentration and equivalency of the base, this loss results in diminished yields and potentially tedious purifications. In a reaction with model dipeptide 1, both reagents produced fully deprotected product and very little or none of the Fmoc amino acid 2 (Table [1], entries 1 and 2). To circumvent these challenges, we sought to optimize the saponification conditions aiming to retain the N-terminal Fmoc group and other base-sensitive functionalities in a peptide building block. Inspired by the prior work,[19] we selected Ca2+ additive in combination with two equivalents of NaOH. Aqueous solution of CaCl2 (0.8 M) was deemed to be a practical reagent that could be scaled up and produced 66% of the desired compound 2 and recovered 1 in 21% (entry 3). Calcium in this reaction plays an important role and other bivalent group 2 metals such as Mg2+ and Ba2+ were either unreactive or led to complete consumption of 1 (entries 4 and 5). We hypothesized that the mixture of BaCl2 and NaOH would produce Ba(OH)2, which would act as the nucleophilic reagent. However, a protocol with one equivalent with Ba(OH)2 or Ca(OH)2 produced only 24% of 2 and almost equimolar amounts of unreacted 1 (entry 7) or a slightly improved yield of 2 (36%, entry 7). Our revised protocol, which employs a combination of NaOH and CaCl2 is operationally simple, yielding 88% of carboxylic acid 2 after 12 h at room temperature, with no recovered starting material (entry 8).[20]

Table 1 Optimization of Fmoc-Compatible Saponification Conditionsa

Entry

Base (equiv.)

Additive (M)

Solvent

Conc. (M)

Time (h)

Yield of 2 (%)

Recovered 1 (%)

1

NaOH (2)

none

i-PrOH/H2O (7:3)

0.04

12

 0

 0

2

LiOH (2.5)

none

THF/H2O (2:1)

0.10

15

11

 0

3

NaOH (2)

CaCl2 (0.8)

i-PrOH/H2O (7:3)

0.04

 5

66

21

4

NaOH (2)

MgCl2 (0.8)

i-PrOH/H2O (7:3)

0.04

12

N.R.

N.R.

5

NaOH (2)

BaCl2 (0.8)

i-PrOH/H2O (7:3)

0.04

12

 0

N.R.

6

Ba(OH)2 (1)

none

i-PrOH/H2O (7:3)

0.04

12

24

21

7

Ca(OH)2 (1)

none

i-PrOH/H2O (7:3)

0.04

12

36

27

8

NaOH (2)

CaCl2 (0.8)

i-PrOH/H2O (7:3)

0.04

12

88

 0

a Reaction yields refer to isolated analytically pure material from a 0.2 mmol scale reaction.

The optimized protocol was next applied to a series of dipeptides with DMB and HMB groups (Scheme [2]). First, we established conditions for the synthesis of a collection of dipeptides using a one-pot reductive amination of glycine ethyl ester 6 with 2,4-dimethoxybenzaldehyde 5a (or 2-hydroxy-4-methoxybenzaldehyde 5b) in the presence of a borohydride reducing agent. The ethyl ester was selected as an inexpensive Gly building block, but the same transformation is also feasible with the methyl derivative. The crude material was then taken to couple with Fmoc-protected amino acids 3 with HATU. The combined yields of these reactions were overall good to excellent, and the dipeptides were isolated in 79–96% yield. The dipeptides were next subjected to the conditions given in Table [1], entry 7, to produce free carboxylic acids in 51–82% yield after chromatographic purification on silica gel (Scheme [3]). The synthesis of DMB protected dipeptides 2 and 7a was completed on a multi-gram scale, highlighting the scalability of the method.

Zoom Image
Scheme 2 Synthesis of DMB- and HMB-protected glycine dipeptides
Zoom Image
Scheme 3 Calcium-mediated ester hydrolysis of glycine dipeptides

Next, we wondered whether the hydrolysis conditions could be applied to pseudoproline dipeptides. We optimized the conditions for the preparation of these valuable building blocks (Scheme [4]). First, the dipeptides were produced in a solution-phase coupling followed by a reaction with 2,2-dimethoxypropane (DMP) and pyridine p-toluenesulfonic acid (PPTS) under reflux. We observed that the yields of these two steps varied greatly between various groups, due to incomplete cyclization of the hemiaminal ether. Similar to the reactions with DMB dipeptides, the calcium mediated conditions resulted in high yields of the free acids 10 (Scheme [5]). The hydrolysis of pseudoproline esters is also scalable, as demonstrated in a reaction with 9f that afforded 1 g of 10f in 64% yield (for details, see the Supporting Information).

Zoom Image
Scheme 4 Synthesis of pseudoproline dipeptides. Yields refer to individual steps or are given over two steps. a The yield of dipeptide after the coupling step. b The yield of pseudoproline ester after the cyclization step.

The unique role of calcium in the basic hydrolysis has been noted before[19] and, mechanistically, these mild conditions can be attributed to both the stabilization of the Fmoc group by Ca2+ and the lability of the ester to Ca(OH)2. Although extended reaction times under the optimized conditions will ultimately lead to the loss of the Fmoc group, the reaction yield can be optimized for an individual substrate, depending on other functional groups in the molecule or the solubility.

In summary, we reported a mild and practical method for the synthesis of free carboxylic acids of dipeptides protected with an Fmoc group at the N-terminal position. This protocol offers an improvement in terms of the overall yield and represents an economical way to access valuable peptide building blocks.

Zoom Image
Scheme 5 Calcium-mediated ester hydrolysis

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Conflict of Interest

The authors declare no conflict of interest.

Acknowledgment

We thank the Central Analytical Mass Spectrometry Facility for their assistance with the spectral analyses.

Supporting Information

    Supporting information for this article is available online at https://doi-org.accesdistant.sorbonne-universite.fr/10.1055/a-2306-9316.
  • Supporting Information

Corresponding Author

Maciej A. Walczak
Department of Chemistry, University of Colorado
Boulder, CO 80309
USA   

Publication History

Received: 05 March 2024

Accepted after revision: 15 April 2024

Accepted Manuscript online:
15 April 2024

Article published online:
21 May 2024

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   Permissions and Reprints All articles of this category
Zoom Image
Scheme 1 Overview of pseudoproline and DMB motifs in peptide synthesis and natural products
Zoom Image
Scheme 2 Synthesis of DMB- and HMB-protected glycine dipeptides
Zoom Image
Scheme 3 Calcium-mediated ester hydrolysis of glycine dipeptides
Zoom Image
Scheme 4 Synthesis of pseudoproline dipeptides. Yields refer to individual steps or are given over two steps. a The yield of dipeptide after the coupling step. b The yield of pseudoproline ester after the cyclization step.
Zoom Image
Scheme 5 Calcium-mediated ester hydrolysis