IChO 2023 · Theory problem 10

Switzerland — The Country of Pharmaceuticals

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Pasireotide (1) is a peptide-based drug developed by the Swiss pharmaceutical company Novartis to treat the Cushing's disease.

Figure 1

10.1 Determine the number of stereogenic centers (\(n\)) in Pasireotide (1). Calculate the total number of all possible stereoisomers (\(t\)).

Pasireotide (1) is a cyclic peptide. An advanced intermediate in its synthesis (linear peptide 2) can be prepared by solid-phase peptide synthesis (SPPS) using the Fmoc/tBu strategy as shown in Scheme 1.

Figure 2

Scheme 1. SPPS of peptide 2. i) Linker; ii) Resin; iii) Resin loading; iv) SPPS: repetition of 1. Fmoc deprotection 2. amino acid coupling + final Fmoc deprotection; v) Peptide cleavage from resin and deprotection of PG-2.

The synthesis starts with the preparation of Fmoc-Tyr(Bn)-OH (3) from Boc-Tyr-OH (7).

Figure 3

10.2 Draw reagents A and D and intermediates B and C in the synthesis of Fmoc-Tyr(Bn)-OH (3) as shown above.

The SPPS of intermediate 2 begins with attaching the Fmoc-Tyr(Bn)-OH (3) to a suitable resin-bound linker.

Figure 4

Scheme 2. ii) Resin; a) 2-Chlorotrityl-chloride linker; b) Safety-catch linker; c) Rink amide linker; d) SASRIN-chloride linker; e) Sieber amide linker; f) Wang linker

10.3 Choose the linker(s) 4 from Scheme 2 that are appropriate for SPPS of peptide 2 according to Scheme 1.

Figure 5

10.4 From g–l choose the most suitable side-chain protecting groups PG-1 and PG-2 for SPPS of 2 according to Scheme 1 that can be orthogonally cleaved in the presence of all other functional groups present in Pasireotide. Only one answer is correct for each of the protecting groups.

Next, linear peptide 2 undergoes an intramolecular coupling reaction to form cyclic peptide 8 according to the following scheme:

Figure 6

Scheme 3. vi) Base

10.5 Choose the correct statement(s) about the cyclization of peptide 2 to 8.

(a) A possible side-product of the reaction is tetramethylguanidylation of the \(N\)-terminal phenylalanine residue resulting in compound 9 shown below.

(b) A possible side-product of the reaction is the cleavage of protecting group PG-1 and cyclization via the amino group of the lysine residue to give compound 10 shown below.

(c) The reaction must be carried out at a high peptide concentration to achieve a sufficient reaction rate.

(d) The reaction must be carried out at a low peptide concentration to prevent polycondensation.

(e) Piperidine (11) below is a suitable base for the reaction.

Figure 7
Figure 8

The last steps of the synthesis involve functionalization of the OH-group of the 4-hydroxyproline residue in 8, followed by cleavage of all PGs to give Pasireotide (1).

Figure 9

Scheme 4. vii) can be used as simplification of 8; viii) Cleavage of protecting groups

10.6 Draw the structures of intermediate E (including stereochemistry) and reagent F. Abbreviate intermediate 8 as (vii) and the protecting group as PG-1 in structures E and F as depicted in Scheme 5.

10.7 Determine the lowest possible molar equivalents of compound 12 that are necessary to enable full conversion of 8 to 13.

Official English text of the problem.

Answers

Asterisks in the SMILES below denote the explicitly identified attachment points to an omitted resin, peptide, or protecting group.

  • 10.1: \(\boxed{n=7,\qquad t=2^7=128}\).

  • 10.2: Suitable reagents and the corresponding intermediates are:

Label Identification SMILES
A Benzyl bromide, used to introduce both benzyl groups BrCc1ccccc1
B \(N\)-Boc-\(O\)-benzyl-L-tyrosine, shown as its free acid after work-up CC(C)(C)OC(=O)N[C@@H](Cc1ccc(OCc2ccccc2)cc1)C(=O)O
C \(O\)-Benzyl-L-tyrosinium trifluoroacetate [NH3+][C@@H](Cc1ccc(OCc2ccccc2)cc1)C(=O)O.O=C([O-])C(F)(F)F
D 9-Fluorenylmethyl chloroformate, Fmoc-Cl O=C(Cl)OCC1c2ccccc2-c2ccccc21

  • 10.3: \(\boxed{\text{a and d}}\): the 2-chlorotrityl-chloride and SASRIN-chloride linkers. Their resin-bound fragments are, respectively, [*:1]c1ccc(C(Cl)(c2ccccc2)c2ccccc2Cl)cc1 and COc1cc(O[*:1])ccc1CCl, where [*:1] represents the resin attachment.

  • 10.4: \(\boxed{\text{PG-1 = i, Boc;\quad PG-2 = l, trityl}}\). The attached protecting-group fragments are tert-butoxycarbonyl, [*]C(=O)OC(C)(C)C, and triphenylmethyl, [*]C(c1ccccc1)(c1ccccc1)c1ccccc1.

  • 10.5: \(\boxed{\text{(a) and (d)}}\).

  • 10.6: E is the 4-\(O\)-chloroformate of peptide 8, retaining the hydroxyproline \((2S,4R)\) configuration: [*:1]N1C[C@H](OC(=O)Cl)C[C@H]1C(=O)[*:2]. The two asterisks are the peptide continuation bonds shown in abbreviation (vii). F is \(N\)-Boc-ethane-1,2-diamine, NCCNC(=O)OC(C)(C)C; with the requested PG-1 abbreviation it is NCCN[*:3], where [*:3] denotes PG-1.

  • 10.7: \(\boxed{\dfrac{1}{3}\text{ molar equivalent of 12}}\), or \(0.333\ \mathrm{mol}\) of triphosgene per \(1.00\ \mathrm{mol}\) of peptide 8.

Solution

10.1 — Stereogenic centers and stereoisomers

Each of the six amino-acid residues has a stereogenic \(\alpha\)-carbon:

  1. Phenylalanine.
  2. 4-Hydroxyproline.
  3. Phenylglycine.
  4. Tryptophan.
  5. Lysine.
  6. \(O\)-Benzyltyrosine.

The oxygen-bearing carbon at position 4 of the hydroxyproline ring supplies one additional stereogenic center. Thus,

\[ n=6+1=\boxed{7}. \]

The amide nitrogens are approximately planar and do not provide additional configurationally stable stereogenic centers. The primary amino groups each have two identical hydrogen substituents.

There is no molecular symmetry that makes different assignments of these seven centers equivalent or creates meso forms. Therefore,

\[ t=2^n=2^7=\boxed{128}. \]

Ordinary conformational changes, including peptide-bond conformations, are not counted separately in this stereogenic-center calculation.

The depicted pasireotide has the stereochemical SMILES:

N1[C@@H](Cc2ccccc2)C(=O)N2C[C@H](OC(=O)NCCN)C[C@H]2C(=O)N[C@@H](c2ccccc2)C(=O)N[C@H](Cc2c[nH]c3ccccc23)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](Cc2ccc(OCc3ccccc3)cc2)C1=O

This specifies connectivity and configuration, but does not specify a particular macrocycle conformation.

10.2 — Preparation of Fmoc-Tyr(Bn)-OH

The sequence protects both oxygen functions with benzyl groups, selectively hydrolyzes the benzyl ester, removes Boc, and installs Fmoc. The tyrosine stereocenter retains its \(S\) configuration throughout.

The structures already drawn in this sequence can be represented as follows:

Compound Name SMILES
7 \(N\)-Boc-L-tyrosine CC(C)(C)OC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)O
Product immediately after A Benzyl \(N\)-Boc-\(O\)-benzyl-L-tyrosinate CC(C)(C)OC(=O)N[C@@H](Cc1ccc(OCc2ccccc2)cc1)C(=O)OCc1ccccc1
3 \(N\)-Fmoc-\(O\)-benzyl-L-tyrosine O=C(O)[C@@H](NC(=O)OCC1c2ccccc2-c2ccccc21)Cc1ccc(OCc2ccccc2)cc1

A: benzylation of both oxygen functions. Potassium carbonate generates the nucleophilic carboxylate and phenoxide. Each attacks benzyl bromide at its benzylic carbon by an \(S_\mathrm{N}2\) substitution. The products are a benzyl ester and a benzyl ether.

Using the amino-acid shorthand of the problem, the balanced overall equation is

\[ \ce{Boc-Tyr-OH + 2 PhCH2Br + K2CO3 -> Boc-Tyr(Bn)-OBn + 2 KBr + CO2 + H2O}. \]

Thus A is benzyl bromide, with two equivalents required for complete dibenzylation. Benzyl chloride is another chemically suitable benzylating reagent.

B: selective ester hydrolysis. Potassium hydroxide hydrolyzes the benzyl ester by nucleophilic acyl substitution. The aryl benzyl ether and the Boc group survive these basic conditions:

\[ \ce{Boc-Tyr(Bn)-OBn + KOH -> Boc-Tyr(Bn)-OK + PhCH2OH}. \]

Here “-OK” denotes the potassium carboxylate. Acidification gives the free acid:

\[ \ce{Boc-Tyr(Bn)-OK + H+ -> Boc-Tyr(Bn)-OH + K+}. \]

Therefore B is \(N\)-Boc-\(O\)-benzyl-L-tyrosine. Strictly, before acidic work-up the alkaline mixture contains its potassium carboxylate, CC(C)(C)OC(=O)N[C@@H](Cc1ccc(OCc2ccccc2)cc1)C(=O)[O-].[K+].

The alcohol released is benzyl alcohol, OCc1ccccc1.

C: removal of Boc. Concentrated trifluoroacetic acid removes the Boc group while preserving the benzyl ether. Boc cleavage gives carbon dioxide and, in a formal net equation, 2-methylpropene. The newly exposed amino group is protonated by the acid.

Let \(R\) denote the tyrosine carbon skeleton attached to nitrogen, including its benzyl-protected phenol and free carboxyl group. Then

\[ \ce{RNHCO2C(CH3)3 + CF3CO2H -> [RNH3]+ + CF3CO2- + CO2 + CH2=C(CH3)2}. \]

Consequently, C is \(O\)-benzyl-L-tyrosinium trifluoroacetate. The relevant small organic species are trifluoroacetic acid, O=C(O)C(F)(F)F, and 2-methylpropene, C=C(C)C.

D: installation of Fmoc. Fmoc-Cl reacts with the liberated amino group by nucleophilic acyl substitution. Bicarbonate releases the amine from its ammonium salt and neutralizes the acid generated during carbamate formation.

Writing \(Fl\) for the fluoren-9-yl group, a balanced equation for conversion of the trifluoroacetate salt to the isolated protected amino acid is

\[ \ce{ [RNH3]+ + CF3CO2- + ClCO2CH2Fl + 2 NaHCO3 -> RNHCO2CH2Fl + NaCF3CO2 + NaCl + 2 CO2 + 2 H2O }. \]

Thus D is Fmoc-Cl, and the product is 3, Fmoc-Tyr(Bn)-OH.

10.3 — Selection of the linker

Two requirements follow directly from Scheme 1:

  • Cleavage must produce the C-terminal carboxylic acid needed for subsequent cyclization.
  • Cleavage must occur with 1% trifluoroacetic acid for 15 minutes, while PG-1 remains attached.
Linker Assessment
a: 2-Chlorotrityl-chloride Suitable. Gives an acid-labile ester linkage and releases the peptide carboxylic acid under mild acidic conditions.
b: Safety-catch Unsuitable. Its intended cleavage requires activation and subsequent cleavage chemistry not provided in Scheme 1.
c: Rink amide Unsuitable. Releases a C-terminal amide rather than the required carboxylic acid.
d: SASRIN-chloride Suitable. Its highly acid-labile ester releases the peptide carboxylic acid under the specified mild conditions.
e: Sieber amide Unsuitable. Although readily acid-cleavable, it gives a C-terminal amide.
f: Wang Unsuitable for the specified conditions. Its ester normally requires substantially stronger acid treatment.

For both selected chloride linkers, the amino-acid carboxylate replaces the linker chloride. Thus the attachment atom \(Y\) in Scheme 1 is oxygen.

\[ \boxed{\text{Linkers a and d}} \]

10.4 — Selection of the protecting groups

PG-1 must be Boc, option i.

The lysine side-chain amino group and the tryptophan indole nitrogen must remain protected during:

  • Repeated basic Fmoc removals.
  • Peptide coupling.
  • The brief, dilute-acid resin cleavage.
  • Macrocyclization and hydroxyproline functionalization.

Boc provides the required compatibility. Its later acid-mediated removal preserves the benzyl ether that is part of the final drug.

The alternatives illustrate the orthogonality constraints. Fmoc would be removed during the repeated Fmoc-deprotection steps. Cbz removal by hydrogenolysis would also threaten the required tyrosine benzyl ether. Trityl would be removed during the mild acid treatment intended to remove PG-2.

PG-2 must be trityl, option l.

The hydroxyproline oxygen must remain protected during SPPS but become a free alcohol during the 1% trifluoroacetic acid treatment. A trityl ether is sufficiently acid-labile for this selective cleavage because the departing triphenylmethyl cation is strongly resonance-stabilized.

The tert-butyl ether is insufficiently labile under the specified brief, dilute-acid conditions; conditions strong enough to remove it would also compromise the desired retention of Boc.

Therefore,

\[ \boxed{\text{PG-1 = Boc (i),\qquad PG-2 = Trt (l)}}. \]

10.5 — Cyclization

The intended reaction joins the terminal amino and carboxyl groups. Its formal condensation balance is

\[ \ce{H2N-P-CO2H -> cyclo(-NH-P-CO-) + H2O}, \]

where \(P\) denotes the intervening peptide skeleton. This expresses the net structural change; the coupling reagent mediates activation and produces its own by-products.

The reagent drawn is HBTU, \(O\)-(benzotriazol-1-yl)-\(N,N,N',N'\)-tetramethyluronium hexafluorophosphate:

CN(C)C(=[N+](C)C)On1nnc2ccccc21.[P-](F)(F)(F)(F)(F)F

Statement Verdict Reason
(a) Correct The terminal amine can attack HBTU’s electrophilic uronium carbon directly, installing a tetramethylguanidino group and giving the side-product connectivity shown in 9.
(b) Incorrect PG-1 is Boc, which is stable under the basic coupling conditions. The lysine side-chain amino group remains protected.
(c) Incorrect High concentration increases intermolecular peptide coupling and therefore favors oligomerization.
(d) Correct Dilution suppresses intermolecular coupling relative to intramolecular cyclization.
(e) Incorrect Piperidine is a nucleophilic secondary amine and can compete with the peptide amino group for the activated carboxyl group.

The tetramethylguanidino substituent has the neutral-form fragment [*]N=C(N(C)C)N(C)C, where the asterisk denotes the peptide \(\alpha\)-carbon attachment; protonation changes its charge distribution without changing this connectivity.

The concentration argument follows from the approximate rate laws

\[ v_{\mathrm{cyclization}}=k_{\mathrm{intra}}[P], \qquad v_{\mathrm{intermolecular}}=k_{\mathrm{inter}}[P]^2. \]

Hence

\[ \frac{v_{\mathrm{intermolecular}}}{v_{\mathrm{cyclization}}} = \frac{k_{\mathrm{inter}}}{k_{\mathrm{intra}}}[P]. \]

Lowering peptide concentration reduces the relative importance of intermolecular reactions.

Piperidine, N1CCCCC1, is therefore unsuitable as the coupling base. A suitable choice is the hindered tertiary amine \(N,N\)-diisopropylethylamine, CCN(C(C)C)C(C)C.

\[ \boxed{\text{Correct statements: (a) and (d)}} \]

10.6 — Intermediate E and reagent F

Compound 12 is triphosgene, or bis(trichloromethyl) carbonate:

O=C(OC(Cl)(Cl)Cl)OC(Cl)(Cl)Cl

It supplies phosgene, O=C(Cl)Cl, equivalents.

Let \(\ce{R-OH}\) denote the protected cyclic peptide 8, with the oxygen belonging to the hydroxyproline hydroxyl group. Conversion to the chloroformate is

\[ \ce{R-OH + COCl2 -> R-O-C(=O)Cl + HCl}. \]

Therefore E is the hydroxyproline \(O\)-chloroformate.

To draw E using abbreviation (vii), replace the hydroxyl hydrogen by \(\ce{C(=O)Cl}\), giving the substituent \(\ce{-O-C(=O)Cl}\). Preserve the dashed bond from hydroxyproline C4 to oxygen and the solid wedge shown in the pyrrolidine ring. The configuration remains \((2S,4R)\).

Its abbreviated stereochemical SMILES is

[*:1]N1C[C@H](OC(=O)Cl)C[C@H]1C(=O)[*:2]

Both asterisks connect to the omitted remainder of the same cyclic peptide.

The side chain in 13 requires one free amino group to attack the chloroformate and a second amino group protected by PG-1. Thus F is mono-Boc-protected ethane-1,2-diamine:

\[ \ce{H2N-CH2-CH2-NH-PG1}. \]

Its abbreviated SMILES is NCCN[*:3], with [*:3] = PG-1; its complete SMILES is NCCNC(=O)OC(C)(C)C.

The free amino group attacks the chloroformate carbonyl, and chloride leaves:

\[ \ce{ R-O-C(=O)Cl + H2N-CH2-CH2-NH-PG1 -> R-O-C(=O)-NH-CH2-CH2-NH-PG1 + HCl }. \]

This gives the carbamate in 13. Neither step breaks a bond at a stereogenic carbon, so the hydroxyproline configurations are retained. A base can capture the hydrogen chloride generated in these reactions.

10.7 — Minimum amount of triphosgene

One molecule of triphosgene contains three carbon atoms that can supply three phosgene equivalents:

\[ \ce{(CCl3O)2CO -> 3 COCl2}. \]

Each conversion of 8 into 13 requires one new carbamate carbonyl. Therefore one mole of triphosgene can, in principle, functionalize three moles of peptide 8.

The overall balanced functionalization equation is

\[ \ce{ 3 R-OH + 3 H2N-CH2-CH2-NH-PG1 + (CCl3O)2CO -> 3 R-O-C(=O)-NH-CH2-CH2-NH-PG1 + 6 HCl }. \]

Thus,

\[ 3n_{12}\geq n_8, \]

and the theoretical minimum is

\[ \boxed{ \left(\frac{n_{12}}{n_8}\right)_{\min} =\frac13 =0.333\ \mathrm{mol\,mol^{-1}} }. \]

This is exactly one-third of a molar equivalent, assuming complete utilization of all three phosgene equivalents.

The solution was checked against the official solution.